Anesth Analg 2001;92:100-105
© 2001 International Anesthesia Research Society
ANESTHETIC PHARMACOLOGY
A Dose-Response Study of Anticholinesterase Drugs on Contractile and Phosphatidylinositol Responses of Rat Trachea
Atsushi Tsuda, MD,
Osamu Shibata, MD,
Masataka Saito, MD,
Shigeru Hashimoto, MD,
Shu Iwanaga, MD,
Tetsuji Makita, MD, and
Koji Sumikawa, MD
Department of Anesthesiology, Nagasaki University School of Medicine, Nagasaki, Japan
Address correspondence and reprint requests to Atsushi Tsuda, MD, Department of Anesthesiology, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan. Address e-mail to d397049x{at}stcc.nagasaki-u.ac.jp
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Abstract
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We investigated whether anticholinesterase drugs in large doses inhibit muscarinic receptors of airway smooth muscle. In vitro measurements of isometric tension and [3H]inositol monophosphate (IP1) that formed were conducted by using rat tracheal rings or slices. Neostigmine and pyridostigmine caused muscular contraction and IP1 accumulation in small doses (10 µM and 
100 µM, respectively), but they attenuated muscular contraction and IP1 accumulation in larger doses (1000 µM). Edrophonium did not affect the smooth muscle tone and IP1 levels. Neostigmine, pyridostigmine, and edrophonium attenuated the carbachol (5.5 µM)-induced smooth muscle contraction and IP1 accumulation, when administered in large doses (1000 µM). The attenuation of contraction by neostigmine at large doses was not affected by methoctramine, an M2 muscarinic receptor antagonist, but was reversed by washing with fresh Krebs-Henseleit solution. The results suggest that anticholinesterase drugs have dual effects on the tension and phosphatidylinositol responses of rat trachea. Large doses of anticholinesterase drugs cause airway smooth muscle relaxation, which may be seen in patients with myasthenia gravis who have received excessive anticholinesterase therapy.
Implications: Neostigmine and pyridostigmine, but not edrophonium, have dual effects on the tension and phosphatidylinositol responses of rat trachea. Large doses of anticholinesterase drugs cause airway smooth muscle relaxation, which may be seen in patients with myasthenia gravis who have received excessive anticholinesterase therapy.
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Introduction
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We have observed previously (1,2) that small doses of neostigmine and pyridostigmine stimulated the muscular contraction and phosphatidylinositol (PI) responses of rat trachea, whereas edrophonium had no effect, and that these responses were inhibited by 4-diphenylacetoxy-N-methyl-piperidine methobromide (4-DAMP), an M3 muscarinic receptor antagonist. Anticholinesterase (anti-ChE) drugs directly activate the M3 receptors in tracheal smooth muscle cell membrane. However, large doses of neostigmine may cause neuromuscular block by direct stimulation of nicotinic receptors (3). This condition is seen in patients with organophosphorous (insecticides) poisoning or in those with myasthenia gravis after an anti-ChE overdose (4). Neostigmine has a dual effect on the sinoatrial node of guinea pig right atria. In smaller doses, it causes bradycardia by stimulating postsynaptic muscarinic receptors, but an antimuscarinic effect is seen in large doses (5). However, no data are available on the effect of large doses of anti-ChE drugs on airway smooth muscle. Thus, our purpose was to examine the effects of various doses of anti-ChE drugs on the contractile and PI responses of the rat trachea.
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Methods
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The studies were conducted under guidelines approved by our animal care committee. Sixty-nine male Wistar rats weighing 250–350 g were used for the experiments. The rats were anesthetized with pentobarbital (50 mg/kg intraperitoneally), and their tracheae were rapidly isolated.
Isometric tensions were measured as described previously (2). 1) Anti-ChE drugs (neostigmine, pyridostigmine, or edrophonium) were added in a stepwise manner to induce active contraction/relaxation at 10- to 1000-µM concentration. After completion of the neostigmine experiment, Krebs-Henseleit solution (mM composition: NaCl 118, KCl 4.7, CaCl2 1.3, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, glucose 11, Na2-EDTA 0.05) containing neostigmine (in the organ chamber) was changed once with fresh Krebs-Henseleit solution and the tension was recorded. 2) Carbachol (5.5-µM final concentration) was added, and 30 min later, ring relaxation was induced by stepwise cumulative additions of neostigmine, pyridostigmine, or edrophonium from 10 to 1000 µM in final concentrations. 3) N
-nitro-L-arginine methyl ester (L-NAME) (1-µM final concentration), indomethacin (10-µM final concentration), propranolol (1-µM final concentration), or glybenclamide (10-µM final concentration) was added, and 30 min later, ring contraction/relaxation was induced by cumulative additions of neostigmine at stepwise concentrations from 10 to 1000 µM (final concentrations). 4) Neostigmine was added in a stepwise manner to induce active contraction/relaxation at 10- to 1000-µM concentration. Fifteen minutes after the addition of 1000 µM of neostigmine, 4-DAMP (0.01- to 100-µM final concentrations), an M3 muscarinic receptor antagonist, or methoctramine (0.01- to 10-µM final concentrations), an M2 muscarinic receptor antagonist, was added.
[3H]inositol monophosphate (IP1) in tracheal slices incubated with [3H]myo-inositol was measured asdescribed previously (1,2). 1) Neostigmine (10- to1000-µM final concentrations), pyridostigmine (10-to 1000-µM final concentrations), or edrophonium(10- to 1000-µM final concentrations) was added, and the tubes were incubated for an additional 60 min. 2) Varying doses (10–1000 µM) of neostigmine, pyridostigmine, and edrophonium were added, and 15 min later, carbachol (5.5-µM final concentration) was added. The tubes were reincubated for an additional 60 min. The reaction was stopped with chloroform/methanol (1:2 v/v). Chloroform and water were then added and the phases were separated by centrifugation at 90g over a period of 5 min. The [3H]IP1 was separated from [3H]myo-inositol by column chromatography. The [3H]IP1 that formed in the tracheal slices was counted with a liquid scintillation counter and measured in becquerels. The scintillation counts of the blank values (no slices present) were subtracted to obtain the experimental data.
Data were expressed as mean ± SE. The results of repeated measures and multiple groups were subjected to two-way analysis of variance. Multiple pairwise comparisons between groups were assessed by Scheffé test. A P value < 0.05 was considered significant.
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Results
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The contraction/relaxation responses of the resting tension of rat tracheal rings to neostigmine, pyridostigmine, and edrophonium are shown in Figure 1A. The resting tension of rat tracheal rings was stimulated by neostigmine at doses of 10 µM, and by pyridostigmine at doses of 100 µM, but it was not affected by edrophonium. However, neostigmine and pyridostigmine contraction was decreased during the course of dose-dependent application. In contrast with neostigmine or pyridostigmine, edrophonium did not affect the tracheal resting tension. The effects of neostigmine, pyridostigmine, or edrophonium on carbachol-induced contraction of rat tracheal rings are shown in Figure 1B. Carbachol-induced tracheal contraction was attenuated by neostigmine, pyridostigmine, or edrophonium at a dose of 1000 µM. The effects of L-NAME, indomethacin, propranolol, or glibenclamide on neostigmine-induced contraction/relaxation of rat tracheal rings are shown in Figure 2, A and B. L-NAME, indomethacin, propranolol, or glibenclamide did not affect the contraction/relaxation effects of neostigmine. The attenuation of contraction by neostigmine at large doses was neither affected by 4-DAMP nor methoctramine. The effects of neostigmine, pyridostigmine, and edrophonium on IP1 accumulation of rat tracheal slices are shown in Figure 3A. IP1 accumulation was stimulated by neostigmine at a dose of 10 µM, and by pyridostigmine at doses of 100 µM, but it was not affected by edrophonium. However, IP1 accumulation by neostigmine or by pyridostigmine decreased at a dose of 1000 µM. The effects of neostigmine, pyridostigmine, or edrophonium on carbachol-induced IP1 accumulation are shown in Figure 3B. Carbachol-induced IP1 accumulation was attenuated by 1000 µM of neostigmine, 1000 µM of pyridostigmine, and 
100 µM of edrophonium. Concentration-effect relationships for neostigmine and pyridostigmine are shown in Figure 4. Decreases in IP1 accumulation (at large doses) were consistent with relaxation of rat tracheal rings. The neostigmine-induced contraction was completely reversed after the compound was washed out (Figure 5).
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Figure 1. A, Effects of neostigmine, pyridostigmine, and edrophonium on the resting tension of rat trachea (mean ± SE, n = 6–8). **P < 0.01 vs 10 µM of neostigmine, #P < 0.0001 vs 10 µM of neostigmine, @P < 0.0001 vs 100 µM of pyridostigmine. B, Effects of neostigmine, pyridostigmine, and edrophonium on the 5.5-µM carbachol-induced tension of rat trachea (mean ± SE, n = 7–8). *P < 0.05, ***P < 0.001 vs 0.
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Figure 2. A, Effects of neostigmine-induced contraction/relaxation of rat trachea in the absence (control) and presence of 10 µM of indomethacin or 1 µM of N-nitro-L-arginine methyl ester (L-NAME) (mean ± SE, n = 8). B, Effects of neostigmine-induced contraction/relaxation of rat trachea in the absence (control) and presence of 10 µM of glibenclamide, or 1 µM of propranolol (mean ± SE, n = 6–8).
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Figure 3. A, Effects of neostigmine, pyridostigmine, and edrophonium on inositol monophosphate (IP1) accumulation of rat trachea (mean ± SE, n = 6–7) *P < 0.05 vs 100 µM of pyridostigmine, **P < 0.01 vs 10 µM of neostigmine. B, Effects of neostigmine, pyridostigmine, and edrophonium on 5.5 µM of carbachol-induced IP1 accumulation of rat trachea (mean ± SE, n = 6–7). *P < 0.05, **P < 0.01, and ***P < 0.001 vs 0.
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Figure 4. Concentration-effect relationship between inositol monophosphate (IP1) accumulation and relaxation of rat trachea. Neostigmine-induced attenuation of IP1 accumulation at large doses is statistically correlated with relaxation of rat tracheal rings (r = 0.947, P < 0.0001; n = 7). Pyridostigmine-induced attenuation of IP1 accumulation at large doses is statistically correlated with relaxation of rat tracheal rings (r = 0.868, P < 0.001; n = 7).
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Figure 5. Effects of washing on neostigmine (Neo)-induced tension of rat tracheal rings (mean ± SE, n = 8). ***P < 0.001.
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Discussion
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The present results show that neostigmine and pyridostigmine, but not edrophonium, have dual effects on the tension and PI responses of rat trachea. With smaller doses, neostigmine and pyridostigmine stimulate contractile and PI responses in a dose-dependent manner (1,2). In the present study, subsequent applications with larger doses of these drugs decreased the contractile and PI responses. Although the mechanism involved in the dual effects of anti-ChE drugs on the tension and PI responses has not been clarified, the following explanations may be advanced: 1) Anti-ChE drugs with larger doses may cause the release of a prostanoid or nitric oxide (NO) from epithelium, resulting in a decrease in airway smooth muscle contraction. NO and prostanoid (prostaglandin E2) play an important regulatory role in airway tone (6–9). NO and prostanoid released from epithelium induce airway relaxation. These suggest that anti-ChE drugs may affect NO or prostanoid release, resulting in a modification of carbachol-induced or anti-ChE drug-induced tracheal contraction.
However, neither L-NAME (inhibitor of NO synthase) nor indomethacin affected the decrease in tracheal contraction by anti-ChE drugs, at large doses. Thus, the decrease in tracheal contraction by anti-ChE drugs could not be attributed to releasing NO or prostanoid. 2) Anti-ChE drugs with larger doses may cause stimulation of ß2-adrenergic receptors or activation of adenosine triphosphate-sensitive potassium channels, resulting in a decrease in airway smooth muscle contraction. Neither propranolol nor glibenclamide affected the decrease in tracheal contraction, however. Thus, the decrease in tracheal contraction by anti-ChE drugs could not be attributed to releasing catecholamines or an activation of adenosine triphosphate-sensitive potassium channels of rat tracheal smooth muscle. 3) Anti-ChE drugs in larger doses may attenuate the release of acetylcholine (ACh). Muscarinic ACh receptors in the airway are divided into M2 and M3 receptors (10,11). M3 receptors exist on airway smooth muscle cell membrane, whereas M2 receptors exist on postganglionic parasympathetic nerve endings.
Stimulation of M3 receptors induces airway smooth muscle contraction through the activation of PI response, whereas stimulation of M2 receptors inhibits ACh release, resulting in attenuation of vagally induced airway smooth muscle contraction. Thus, anti-ChE drugs, at larger doses, may attenuate ACh release from postganglionic parasympathetic nerve endings through the stimulation of M2 receptors, resulting in attenuation of contractile and PI responses in rat trachea. Because parasympathetic postganglionic neurons are close to the targeted end organ, the tracheal rings used in this study would contain parasympathetic postganglionic neurons. However, 1,1-dimethyl-4-phenylpiperazinium iodide, a selective ganglionic nicotinic agonist, does not cause contraction of rat trachea (2). The tracheal tissue we used does not appear to contain a sufficient number of functional postganglionic cells and nerve endings. The attenuation of contraction by neostigmine at large doses was not affected by methoctramine, an M2 muscarinic receptor antagonist. Thus, it is unlikely that the decrease in the carbachol-induced or anti-ChE drug-induced contraction of rat trachea is attributable to inhibition of ACh release. 4) Anti-ChE drugs, at larger doses, may inhibit the M3 muscarinic receptors of airway smooth muscle. Because neostigmine, at larger doses, exhibits an antimuscarinic effect on M2 receptors of the sinoatrial node cells of guinea pig right atria (5), anti-ChE drugs, at larger doses, would also exhibit an antimuscarinic effect on M3 receptors of tracheal smooth muscle, resulting in the attenuation of contractile and PI responses.
Neostigmine and pyridostigmine, in smaller doses, stimulated, and in larger doses, decreased contractile and PI responses of rat trachea in this study. Thus, these results may imply that anti-ChE drugs have two binding sites on the muscarinic M3 receptor of tracheal smooth muscle. The muscarinic receptor has a classical (orthosteric)-binding site and an allosteric-binding site. The activation of the allosteric-binding site changes association, dissociation and equilibrium binding of agonists, and it affects the function of the classical-binding site (12–15). Endou et al. (5) suggested that neostigmine possesses two binding sites on the cardiac muscarinic M2 receptor, and that the binding site with high affinity (orthosteric site) for neostigmine would mediate a negative chronotropic effect of the agent, and the binding is too tight to be dissociated by washing. However, the binding site with low affinity (allosteric site) would mediate the antagonistic effect of neostigmine, and the binding is easily dissociated (5). In our present study, attenuation of tracheal contraction by larger doses of anti-ChE drugs was reversed by washing. Thus, anti-ChE drugs, at smaller doses, would bind strongly to the orthosteric site of M3 muscarinic receptors, resulting in tracheal smooth muscle contraction through the activation of a PI response. In contrast, anti-ChE drugs, at larger doses, would bind to the allosteric site, which inhibits the action of the orthosteric site of M3 muscarinic receptors, resulting in the attenuation of contraction through PI response inhibition. Furthermore, allosteric sites can affect the G-protein-mediated functional responses of muscarinic receptors (14). Electrophysiologic experiments of smooth muscle and radioligand binding studies will be needed to clarify the exact mechanisms of neostigmine- or pyridostigmine-induced dual effects.
Although edrophonium did not affect the resting tension and basal IP1 accumulation of rat trachea in our study, at larger doses, it attenuated the carbachol-induced contraction and IP1 accumulation. Yost and Maestrone (3) showed that large doses of neostigmine and edrophonium resulted in dose-dependent inhibition of nicotinic ACh receptors. Edrophonium at larger doses would bind to the allosteric site, which inhibits the action of the orthosteric site of M3 muscarinic receptors, resulting in the attenuation of carbachol-induced contraction through the inhibition of PI response.
There was a discrepancy between the resting tension and IP1 level in the effects of pyridostigmine. Pyridostigmine at 1000 µM reversed completely the pyridostigmine-induced tracheal contraction, but it did not reverse to basal level on pyridostigmine-induced IP1 accumulation. This suggests that pyridostigmine affected any sites between an increase in PI levels and muscle contraction.
Application with larger doses of anti-ChE drugs decreased the contractile and PI responses of rat trachea. When large doses of anti-ChE drugs are used to reverse the excessive application of nondepolarizing muscle relaxants in the operating room, nicotinic receptors in the neuromuscular junction are inhibited, resulting in muscle relaxants, whereas muscarinic receptors in airway smooth muscle may be inhibited, resulting in inhibition of airway contraction. Inhibition of airway contraction may also be seen in patients with myasthenia gravis who have received excessive anti-ChE therapy.
In conclusion, contraction and IP1 accumulation were stimulated by neostigmine and pyridostigmine at smaller doses, but were inhibited by these drugs at larger doses. The neostigmine-induced contraction was completely reversed after the compound was washed out. These results suggest that neostigmine and pyridostigmine have dual effects on rat tracheal contraction.
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Acknowledgments
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This work was supported, in part, by Grants-in-Aid for Scientific Research C, 10671421, from the Ministry of Education, Science, and Culture, Japan.
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Footnotes
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Presented, in part, at the American Society of Anesthesiologists annual meeting, Orlando, FL, October 1998.
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References
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|---|
-
Shibata O, Kanairo M, Zhang S, et al. Anticholinesterase drugs stimulate phosphatidylinositol response in rat tracheal slices. Anesth Analg 1996; 82: 1211–4.[Abstract]
-
Shibata O, Tsuda A, Makita T, et al. Contractile and phosphatidylinositol responses of rat trachea to anticholinesterase drugs. Can J Anaesth 1998; 45: 1190–5.[Web of Science][Medline]
-
Yost CS, Maestrone E. Clinical concentrations of edrophonium enhance desensitization of the nicotinic acetylcholine receptor. Anesth Analg 1994; 78: 520–6.[Abstract/Free Full Text]
-
Stoelting RK. Pharmacology and physiology in anesthetic practice: anticholinesterase drugs and cholinergic agonists. 3rd ed. Philadelphia: Lippincott-Raven, 1999.
-
Endou M, Tanito Y, Okumura F. A comparison between chronotropic effects of neostigmine and edrophonium in isolated guinea pig right atrium. J Pharmacol Exp Ther 1997; 282: 1480–6.[Abstract/Free Full Text]
-
D’Agostino B, Matera MG, Amorena M, et al. Nitric oxide regulatory role in sensitized guinea pig trachea. Life Sci 1997; 60: PL353–7.
-
Yoshihara S, Nadel JA, Figini M, et al. Endogenous nitric oxide inhibits bronchoconstriction induced by cold-air inhalation in guinea pigs: role of kinins. Am J Respir Crit Care Med 1998; 157: 547–52.[Abstract/Free Full Text]
-
Knight DA, Stewart GA, Thompson PJ. Prostaglandin E2, but not prostacyclin inhibits histamine-induced contraction of human bronchial smooth muscle. Eur J Pharmacol 1995; 272: 13–9.[Web of Science][Medline]
-
Park KW, Dai HB, Lowenstein E, et al. Isoflurane- and halothane-mediated dilation of distal bronchi in the rat depends on the epithelium. Anesthesiology 1997; 86: 1078–87.[Web of Science][Medline]
-
Fryer AD, Maclagan J. Muscarinic inhibitory receptors in pulmonary parasympathetic nerves in the guinea-pig. Br J Pharmacol 1984; 83: 973–8.[Web of Science][Medline]
-
Roffel AF, Elzinga CR, Van Amsterdam RG, et al. Muscarinic M2 receptors in bovine tracheal smooth muscle: discrepancies between binding and function. Eur J Pharmacol 1988; 153: 73–82.[Web of Science][Medline]
-
Ellis J, Huyler J, Brann MR. Allosteric regulation of cloned m1–m5 muscarinic receptor subtypes. Biochem Pharmacol 1991; 42: 1927–32.[Web of Science][Medline]
-
Lee NH, el-Fakahany EE. Allosteric interactions at the m1, m2 and m3 muscarinic receptor subtypes. J Pharmacol Exp Ther 1991; 256: 468–79.[Abstract/Free Full Text]
-
Jakubik J, Bacakova L, Lisa V, et al. Activation of muscarinic acetylcholine receptors via their allosteric binding sites. Proc Natl Acad Sci USA 1996; 93: 8705–9.[Abstract/Free Full Text]
-
Christopoulos A, Lanzafame A, Mitchelson F. Allosteric interactions at muscarinic cholinoceptors. Clin Exp Pharmacol Physiol 1998; 25: 185–94.[Web of Science][Medline]
Accepted for publication August 17, 2000.
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Abstract
Introduction
