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ANESTHETIC PHARMACOLOGY

Anticholinesterase Drugs Stimulate Smooth Muscle Contraction of the Rat Trachea Through the Rho-Kinase Pathway

Department of Anesthesiology, Nagasaki University School of Medicine, Nagasaki, Japan

Address correspondence and reprint requests to Osamu Shibata, MD, Department of Anesthesiology, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan. Address e-mail to opshiba{at}net.nagasaki-u.ac.jp.

	Abstract

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We performed this study to determine the effects of Rho-kinase inhibitors, Y-27632 and fasudil, on the anticholinesterase (anti-ChE)-induced contractile and phosphatidylinositol responses of the rat trachea. In vitro measurements of isometric tension and [³H] inositol monophosphate (IP₁) that was formed were conducted by using rat tracheal rings or slices. Neostigmine- and pyridostigmine-induced contractions were almost completely inhibited by Y-27632 and fasudil at 30 µM each, whereas acetylcholine-induced contraction was inhibited incompletely, i.e., by 56% by Y-27632 and by 51% by fasudil, at 100 µM for each, respectively. The inhibitory effects of fasudil on neostigmine- and acetylcholine-induced contractions were completely reversed by calyculin-A, a myosin phosphatase inhibitor. Neostigmine-induced IP₁ accumulation was attenuated by fasudil at 100 µM. The results suggest that anti-ChEs cause airway smooth muscle contraction, in part, through activation of the Rho-kinase pathway.

	Introduction

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Neostigmine (Neo) acts as a cholinergic agonist at cholinergic sites in skeletal muscle, autonomic ganglia, and smooth muscle (1). We previously found that Neo and pyridostigmine (Pyr) stimulate contractile and phosphatidylinositol (PI) responses in rat trachea, whereas edrophonium has no effect (2–4), and that these responses are inhibited by 4-DAMP, a muscarinic M₃ receptor antagonist (3). The findings suggest that anticholinesterase (anti-ChE) drugs directly activate muscarinic M₃ receptors on airway smooth muscle cell membranes.

Airway smooth muscle contraction is regulated by myosin light chain (MLC) phosphorylation (Fig. 1). When muscarinic M₃ receptors on the airway smooth muscle cell membranes stimulate G_q-proteins to activate phospholipase C, inositol 1,4,5 triphosphate (IP₃) is increased. IP₃ mobilizes Ca²⁺ from the sarcoplasmic reticulum, and at the same time Ca²⁺ flows inward from the extracellular space, resulting in an increase in intracellular Ca²⁺ concentration. The increase in Ca²⁺ activates MLC kinase, resulting in an increase in MLC phosphorylation. On the other hand, when receptors on the airway smooth muscle stimulate heterotrimeric G-proteins, and subsequently Rho (small G-proteins), the Rho-kinase pathway is activated and then myosin phosphatase is inactivated, resulting in an increase in MLC phosphorylation (5–8).

View larger version (27K): [in this window] [in a new window]   Figure 1. A flow diagram of the phosphatidylinositol (PI) response and Rho-kinase pathway. G, G-protein; PI, phosphatidylinositol; PIP, phosphatidylinositol 4-phosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; IP3, inositol 1,4,5 trisphosphate; IP2, inositol bisphosphate; IP1, inositol monophosphate; MLC, myosin light chain; P-MLC, phosphorylated myosin light chain.

In the preliminary study, contraction induced by acetylcholine (ACh) reaches a plateau within 5 min, while contractions induced by Neo and Pyr gradually increase and reach a plateau after 30 min (Fig. 2). ACh-induced contraction is terminated by washing, whereas Neo- and Pyr-induced contractions are not terminated even after repeated washings (2). This suggests that a different mechanism might be responsible for the actions of ACh and anti-ChE drugs on the airway smooth muscle. Although anti-ChE drugs directly act on the airway smooth muscle, there are no data regarding the effects of anti-ChE drugs on the Rho-kinase pathway. This study was performed to determine the effects of the Rho-kinase inhibitors, Y-27632 and fasudil, on the airway smooth muscle contraction induced by anti-ChE drugs.

View larger version (18K): [in this window] [in a new window]   Figure 2. A, Typical recordings of neostigmine- and acetylcholine-induced contractions of the rat trachea. B, Effects of acetylcholine and neostigmine on resting tension of the rat trachea (mean ± sd). Acetylcholine, 10 µM; neostigmine, 1 µM.

	Methods

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This study was conducted following guidelines approved by the Animal Care Committee of Nagasaki University. Fifty-seven male Wistar rats (Charles River, Yokohama, Japan) weighing 250–350 g were used for the experiments. The rats were anesthetized with pentobarbital (50 mg/kg intraperitoneally), and their tracheas were rapidly isolated.

Each trachea was cut into 3-mm-wide ring segments with a McIlwain tissue chopper (Mickle Laboratory Engineering, Gomshall, UK). We used the distal part of the trachea. The tracheal ring was suspended between 2 stainless steel hooks and placed in a 5-mL water-jacketed organ chamber (Kishimotoika, Kyoto, Japan) containing Krebs-Henseleit (K-H) 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). The solution was continuously aerated with O₂ 95%/CO₂ 5% at 37°C. Isometric tensions were measured using an isometric transducer (Kishimotoika, Kyoto, Japan) and changes in isometric force were recorded using a MacLab system (Milford, MA). The resting tension was periodically adjusted to 1.0 g during the equilibration period. The rings were washed every 15 min and re-equilibrated to baseline tension for 60 min (Time 0).

To examine the effects of Rho-kinase inhibitors on the ACh-, Neo-, Pyr-, or KCl-induced contraction of rat tracheal rings, 1 µM Neo, 10 µM ACh, 10 µM Pyr, or 40 mM KCl in a final concentration was added. In the present study, Neo was used at a concentration of 1 µM, which was the appropriate ED₅₀ for the contractile response (3). It was observed that Neo- and ACh-induced contractions were sustained over 90 min (Fig. 2A, -B). The concentrations of Neo, ACh, Pyr, and KCl used in the experiment had nearly equal potencies to induce the contractile response. Thirty minutes after application, one of the Rho-kinase inhibitors, Y-27632 and fasudil, was added cumulatively in stepwise concentrations ranging from 1 to 100 µM.

To examine the effects of myosin phosphatase inhibitor on the attenuation by Rho-kinase inhibitor of Neo- or ACh-induced contraction, 1 µM Neo or 10 µM ACh in a final concentration was added, and 30 min later, ID₅₀of fasudil (2.8 µM) for Neo and 100 µM (nearly equal potencies of ID₅₀) for ACh was added. Calyculin A, a myosin phosphatase inhibitor, 1 µM in a final concentration, was added 15 min later and the response was observed for 30 min.

The technique of Brown et al. (9) was used to measure PI response. IP₃ is rapidly degraded into inositol monophosphate (IP₁) and subsequently recycled back to phosphatidylinositol via free inositol. Lithium inhibits the conversion of IP₁ to inositol, and in the presence of Li⁺ the accumulation rate of IP₁ reflects the extent of PI response (Fig. 1). We measured [³H]IP₁ in the tracheal slices incubated with [³H]myo-inositol (Amersham, Tokyo, Japan). Each trachea was longitudinally cut and chopped into 1-mm-wide slices with a McIlwain tissue chopper (Campden Instruments, Leicester, UK). Three pieces of the tracheal slice were placed in small flat-bottomed tubes and preincubated for 15 min in K-H solution containing 5 mM LiCl and continuously aerated with O₂ 95%/CO₂ 5%. An aliquot of 0.5 µCi [³H]myo-inositol was then added to each tube (final concentration of 0.1 µM in a 300 µL incubation volume). The tubes were then flushed with O₂95%/CO₂5%, capped, set in a shaking bath at 37°C and incubated for 30 min (Time 0).

To examine the effects of fasudil on Neo-induced IP₁ accumulation on the rat tracheal pieces, varying doses (1 µM-100 µM) of fasudil were added at Time 0; 1 µM Neo was added 15 min later, and the tubes were re-aerated with O₂95%/CO₂5%, recapped, and reincubated. After an additional 60 min, the reaction was stopped with 940 µL of chloroform: methanol (1:2 v/v). Chloroform and water were then added (310 µL each) and the phases were separated by centrifugation at 90g for 5 min. The [³H]IP₁ was separated from [³H]myo-inositol in the 750 µL water phase by column chromatography using Dowex AG 1-x8 resin (Bio Rad, Richmond, CA) in its formate form. The [³H]IP₁ that formed in the tracheal slices was counted using a liquid scintillation counter and measured in becquerels. The scintillation counts for the blank values (no slices present) were subtracted to obtain the experimental data.

Data are expressed as mean ± sd. The results were subjected to one-way analysis of variance followed by Scheffe’s F-test. A P value < 0.05 was considered significant.

	Results

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The recording of the effects of Y-27632 on Neo-induced contraction of rat tracheal rings is shown in Figure 3A. Figure 3B shows the effects of Y-27632 on ACh-, Neo- Pyr-, and KCl-induced contractions. The tensions induced by Neo, Pyr, and ACh were 1.64 ± 0.54, 1.35 ± 0.67, and 1.38 ± 0.44 g, respectively. There were no significant differences among the contractions induced by 10 µM ACh, 1 µM Neo, and 10 µM Pyr at 30 min after their application. Neo- and Pyr-induced contractions were almost completely inhibited by Y-27632 at 30 µM, whereas ACh-induced contraction was inhibited by 56% by Y-27632 at 100 µM. KCl-induced contraction was inhibited by 57% by Y-27632 at 100 µM. The ID₅₀ values for Y-27632 on Neo- and Pyr-induced tracheal ring contractions were 4.0 ± 3.4 and 4.2 ± 5.7 µM, respectively. Figure 3C shows the effects of fasudil on ACh-, Neo-, Pyr-, and KCl-induced contractions. Neo- and Pyr-induced contractions were almost completely inhibited by fasudil at 30 µM, whereas ACh-induced contraction was inhibited by 51% by fasudil at 100 µM. KCl-induced contraction was inhibited by 76% by fasudil at 100 µM. The ID₅₀ values for fasudil on Neo- and Pyr-induced tracheal ring contractions were 2.8 ± 2.9 and 4.0 ± 4.2 µM, respectively. The recordings of the effects of calyculin-A on the attenuation by fasudil of ACh- and Neo-induced contractions of rat tracheal rings are shown in Figure 4A and 4B. The attenuation of ACh- and Neo-induced contractions by fasudil was reversed by calyculin-A (Fig. 5).

View larger version (25K): [in this window] [in a new window]   Figure 3. A, A typical recording of the effects of Y-27632 on neostigmine-induced contraction of the rat trachea. B, Effects of Y-27632 on acetylcholine-, neostigmine-, pyridostigmine-, and KCl-induced contractions of the rat trachea (mean ± sd). *P < 0.05; **P < 0.01; ***P < 0.001 versus Y-27632 0. Acetylcholine, 10 µM; neostigmine, 1 µM; pyridostigmine, 10 µM; KCl, 40 mM. C, Effects of fasudil on acetylcholine-, neostigmine-, pyridostigmine-, and KCl-induced contractions of the rat trachea (mean ± sd). *P < 0.05; **P < 0.01; ***P < 0.001 versus fasudil 0. @P < 0.01 versus acetylcholine. Acetylcholine, 10 µM; neostigmine, 1 µM; pyridostigmine, 10 µM; KCl, 40 mM.

View larger version (22K): [in this window] [in a new window]   Figure 4. A typical recording of the effects of calyculin-A on attenuation by fasudil of ACh- and Neo-induced contraction of the rat trachea.

View larger version (31K): [in this window] [in a new window]   Figure 5. Effects of calyculin-A on attenuation by fasudil of acetylcholine- or neostigmine-induced contraction of the rat trachea (mean ± sd; n = 7–10). *P < 0.05; ***P < 0.001 versus ACh or Neo; @P < 0.001 versus fasudil; $P < 0.01 versus fasudil; #P < 0.05 versus ACh. Calyculin-A, 1 µM; ACh, 10 µM acetylcholine; Neo, 1 µM neostigmine.

The effect of fasudil on Neo-induced IP₁ accumulation in rat tracheal slices is shown in Figure 6. IP₁ accumulation was stimulated by Neo 1 µM, and this was attenuated by fasudil at 100 µM.

View larger version (23K): [in this window] [in a new window]   Figure 6. Effects of fasudil on neostigmine-induced IP1 accumulation of the rat trachea (mean ± sd; n = 6–11). ***P < 0.001 versus basal; # P < 0.05 versus basal; @P < 0.05 versus fasudil 0. Neostigmine, 1 µM; IP1, inositol monophosphate; Bq, becquerel.

	Discussion

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The present results show that ACh, Neo, and Pyr induce contraction of the rat trachea and that Neo- and Pyr-induced contractions are inhibited by Rho-kinase inhibitors at smaller concentrations in comparison with ACh-induced contraction. The results also show that Neo-induced PI response is inhibited by the Rho-kinase inhibitor at a larger concentration.

When agonists stimulate receptors on airway smooth muscle cell membranes, G_q- and heterotrimeric G-proteins activate the PI response and the Rho-kinase pathway, respectively, resulting in airway smooth muscle contractions. In the present study, fasudil fully affected the contraction but not the PI response, suggesting that fasudil has a selective action on the Rho-kinase pathway. The airway smooth muscle contraction occurs through activation of the receptors coupled with small G-proteins in canine, rabbit, and human airway smooth muscles in vitro and involves the Rho-kinase pathway (6–8). Rho, the small G-protein activates Rho-kinase, which in turn inactivates myosin phosphatase. Inactivation of myosin phosphatase increases MLC phosphorylation, resulting in an increased contraction. In the present study, we examined the role of the Rho-kinase pathway in the effects of Neo and Pyr using Y-27632 and fasudil, and found that the Rho-kinase inhibitors completely inhibit the anti-ChE-induced contraction in rat tracheal rings. Thus, anti-ChE drugs probably in part cause airway smooth muscle contraction through activation of the Rho-kinase pathway.

Chiba et al. (10) examined the effects of Y-27632 on ACh-induced contraction of the bronchial smooth muscle in rats. They found that in ACh-precontracted muscles, maximal relaxation (50% inhibition of contraction) is obtained by Y-27632 at 100 µM, which has no effect on the resting tone. The magnitude of inhibition by Y-27632 or fasudil in the present study is consistent with their results. Thus, although the magnitude of contraction induced by ACh is smaller than that by Neo and Pyr, ACh would at least in part cause the airway smooth muscle contraction through activation of the Rho-kinase pathway.

Rho-kinase inhibitors may attenuate ACh release from postganglionic parasympathetic nerve endings, resulting in attenuation of the anti-ChE drug-induced contractile response in rat trachea. In our previous study, the selective ganglionic nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium iodide, did not cause contraction of the rat trachea (3). However, 1,1-dimethyl-4-phenylpiperazinium iodide is only a weak nicotinic agonist and this observation cannot exclude the possibility that the anti-ChE drugs release ACh from post-ganglionic nerves. Riker and Wescoe (11) reported that Neo induces skeletal muscle contraction in the cat even after complete inactivation of acetylcholinesterase. Sherby et al. (12) observed that anti-ChE drugs bind to nicotinic ACh receptors in rat skeletal muscle and that Neo and Pyr act as partial agonists. In addition, Carlyle (13) showed that Neo produces an atropine-sensitive contraction in guinea pig tracheal muscle, which is abolished by maneuvers that inhibit the release of ACh from nerve terminals. It was observed that Neo-induced contraction was gradually increased and sustained over 90 minutes if no drugs were added (Fig. 2). Thus, because of the known anti-ChE activity, it is possible that in this airway preparation Neo is allowing for the accumulation of released ACh from the postganglionic nerve terminals that accounts for an ACh effect at muscarinic receptors. On the other hand, the Rho/Rho-kinase pathway plays a role not only in smooth muscle contraction but also in the regulation of neurotransmitter release. Buyukafsar and Levent (14) observed ACh release upon electrical field stimulation of the gastric fundus and found that Y-27632 significantly inhibited this release. They concluded that the Rho/Rho-kinase pathway might play a role in ACh release in the mouse. Therefore, Neo- and Pyr-induced contraction might be inhibited by Rho-kinase inhibitors through the inhibition of ACh release from postganglionic parasympathetic nerve endings.

The possible intracellular mechanisms involved in anti-ChE drugs-induced contraction are as follows: Anti-ChE drugs activate muscarinic M₃ receptors and subsequently increase the PI response, resulting in the increase in the intracellular Ca²⁺ concentration. At the same time anti-ChE drugs activate Rho-kinase pathways through the activation of muscarinic M₃ receptors, resulting in an inactivation of myosin phosphatase and subsequent increase in MLC phosphorylation. The increase in MLC phosphorylation would cause contraction of airway smooth muscle.

In the present study, the effective concentration of fasudil for inhibiting Neo-induced contraction of rat tracheal rings was 10 µM (P < 0.01). The peak plasma concentration of fasudil is approximately 0.6 µM in clinical settings (15). Although the concentration required for tracheal smooth muscle relaxation in the present study is out of the clinical range, these results suggest a benefit of these drugs when used to treat patients with anti-ChE drug-induced bronchoconstriction.

In conclusion, anti-ChE drugs cause airway smooth muscle contraction in part through activation of the Rho-kinase pathway.

	Footnotes

 
Supported, in part, by Grant 15591638 for Scientific Research from the Ministry of Education, Science and Culture, Japan.

Accepted for publication November 4, 2005.

Presented, in part, at the 2004 ASA Annual Meeting, Las Vegas, Nevada, October 23–27, 2004.

	References

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Shibata, O. Articles by Sumikawa, K. Search for Related Content PubMed PubMed Citation Articles by Shibata, O. Articles by Sumikawa, K. Related Collections Airway Pharmacology