Anesth Analg 2003;97:1059-1063
© 2003 International Anesthesia Research Society
ANESTHETIC PHARMACOLOGY
Interactions of Edrophonium with Neostigmine in the Rat Trachea
Osamu Shibata, MD,
Masataka Saito, MD,
Maki Yoshimura, MD,
Masakazu Yamaguchi, MD,
Tetsuji Makita, MD, and
Koji Sumikawa, MD
Department of Anesthesiology, Nagasaki University School of Medicine, Nagasaki, Japan
Address correspondence and reprints 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
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Abstract
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The muscarinic M3 receptor of airway smooth muscle has both an orthosteric binding site and an allosteric binding site. Edrophonium may bind to the allosteric site, resulting in the inhibition of the action of the orthosteric site. Therefore, we examined the effects of edrophonium on neostigmine-induced contractile and phosphatidylinositol responses of rat trachea. Neostigmine (100 µM in final concentration) was added, and ring tension was examined by the addition of edrophonium. After the completion of the experiment, Krebs-Henseleit (K-H) solution containing both edrophonium and neostigmine was changed three times with fresh K-H solution, and the tension was recorded. Tracheal slices were incubated with [3H]myo-inositol and 100 µM neostigmine in the presence or absence of edrophonium. The [3H]inositol monophosphate (IP1) was measured. Data were expressed as mean ± SE. Statistical significance (P < 0.05) was determined with analysis of variance. Neostigmine-induced tension and IP1 accumulation were attenuated by edrophonium at concentrations of 100 µM or more. This attenuation was reversed to more than 80% of control levels by washing with fresh K-H solution. The results suggest that edrophonium would bind to the allosteric site, resulting in the inhibition of the action of the orthosteric site of muscarinic M3 receptors of rat trachea.
IMPLICATIONS: We examined the effects of edrophonium on neostigmine-induced contractile and phosphatidylinositol responses of rat trachea. Neostigmine-induced tension and inositol monophosphate accumulation were attenuated by edrophonium. This attenuation was reversed by washing. The results suggest that edrophonium would bind to the allosteric site.
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Introduction
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The muscarinic M3 receptor in airway smooth muscle has both a classic (orthosteric) binding site and an allosteric binding site. Neostigmine at smaller concentrations binds to the orthosteric site of muscarinic M3 receptors, resulting in tracheal smooth muscle contraction through the activation of the phosphatidylinositol (PI) response (1). Neostigmine at larger concentrations binds to the allosteric site, which inhibits the action of the orthosteric site of muscarinic M3 receptors, resulting in the attenuation of contraction through the inhibition of the PI response (Fig. 1) (1). Thus, neostigmine has a dual effect on the muscarinic M3 receptors of rat trachea. However, edrophonium at both smaller and larger concentrations does not affect the resting tension and basal PI response of rat trachea (1–3). In denervated cat heart, in myocardium the predominant receptor subtype is M2, and myocardium does not contain M3 receptors (4). Edrophonium at larger concentrations produces an increase in heart rate in the denervated cat heart (4). Thus, edrophonium at larger concentrations may bind to the allosteric site, resulting in the inhibition of the action of the orthosteric site of muscarinic M3 receptors. Therefore, we examined the effects of edrophonium on neostigmine-induced contractile and PI responses of rat trachea.
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Methods
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The studies were conducted under guidelines approved by the Animal Care Committee of Nagasaki University. Forty 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.
The trachea was cut into 3-mm-wide ring segments with a McIlwain tissue chopper (Mickle Laboratory Engineering, Gomshall, UK). The tracheal ring was suspended between two stainless-steel hooks and placed in a 5-mL water-jacketed organ chamber (Kishimotoika, Kyoto Japan) containing Krebs-Henseleit (K-H) solution (composition [mM]: 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 95% oxygen/5% CO2, with pH maintained at approximately 7.4 and temperature at 37°C. Isometric tensions were measured by using an isometric transducer (Kishimotoika), and changes in isometric force were recorded by using a MacLab system (Milford, MA). The resting tension was adjusted periodically to 1.5 g during the equilibration period. The ring was washed every 15 min and reequilibrated to baseline tension for 60 min (Time 0).
First, tracheal rings were washed with fresh K-H solution to test whether neostigmine binds tightly to the orthosteric-binding site of muscarinic M3 receptors. At Time 0, neostigmine was added in a stepwise fashion to induce active contraction/relaxation at 100–1000 µM concentrations. After complete relaxation, K-H solution containing neostigmine (in the organ chamber) was replaced five times with fresh K-H solution, and the tension was recorded (Fig. 2). Second, the effects of edrophonium on neostigmine-induced contraction of rat tracheal rings were examined. At Time 0, neostigmine (100 µM in final concentration) was added, and 30 min later, edrophonium was added stepwise cumulatively from 1 to 1000 µM (in final concentrations). Third, to examine the effects of washing on edrophonium-induced relaxation, neostigmine (100 µM in final concentration) was added, and 30 min later, edrophonium 100 µM (in final concentration) was added. Ten minutes later, the tracheal ring was washed three times with fresh K-H solution.
Inositol 1,4,5-trisphosphate is rapidly degraded into inositol monophosphate (IP1), which is recycled back to PI via free inositol. Lithium inhibits the conversion of IP1 to inositol. Thus, in the presence of lithium, the accumulation rate of IP1 reflects the extent of PI response. We measured [3H]IP1 in tracheal slices incubated with [3H]myo-inositol (Amersham, Tokyo, Japan). The trachea was cut longitudinally and chopped into 1-mm-wide pieces with a McIlwain tissue chopper. 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. The solution was continuously aerated with 95% oxygen/5% CO2. An aliquot of 0.5 µCi [3H]myo-inositol was then added to each tube (final concentration 0.1 µM in a 300-µL incubation volume), and the tubes were flushed with 95% oxygen/5% CO2, capped, set in a shaking bath at 37°C, and incubated for 30 min (Time 0).
First, the effects of edrophonium on the neostigmine-induced IP1 accumulation of rat tracheal pieces were examined. At Time 0, varying doses (1–100 µM) of edrophonium were added, and 15 min later, neostigmine (100 µM in final concentration) was added. The tubes were reaerated with 95% oxygen/5% CO2, recapped, and then reincubated. After an additional 60 min, the reaction was stopped with 940 µL of chloroform/methanol (1:2 vol/vol). Chloroform and water were then added (310 µL each), and the phases were separated by centrifugation at 90g over 5 min. The [3H]IP1 was separated from [3H]myo-inositol in the 750-µL water phase by column chromatography with Dowex AG 1-X8 resin (Bio-Rad, Richmond, CA) in the formate form. Second, the effects of washing on edrophonium’s attenuation of neostigmine-induced IP1 accumulation of rat tracheal pieces were examined. At Time 0, both edrophonium and neostigmine (100 µM each) were added, and 15 min later the tracheal slices were taken out, washed, wiped, and put into fresh K-H solution containing 0.5 µCi of [3H]myo-inositol. The tubes were reaerated with 95% oxygen/5% CO2, recapped, and reincubated for a further 45 min. The reaction was stopped with 940 µL of chloroform/methanol (1:2 vol/vol), followed by the same procedure as described previously. The [3H]IP1 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 are expressed as mean ± SE. The results were subjected to one-way analysis of variance. Comparisons between groups were assessed by the Scheffé F test. A P value <0.05 was considered significant.
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Results
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The recording of the effects of washing on neostigmine-induced tension of rat tracheal rings is shown in Figure 2. Tension induced by neostigmine at a concentration of 1000 µM was stimulated by washing, and the contraction continued over 2 h. Relaxed responses of neostigmine-induced contraction of rat tracheal rings to edrophonium are shown in Figure 3. The resting tension of rat tracheal rings was stimulated by neostigmine at a concentration of 100 µM and was attenuated by edrophonium at concentrations of 100 µM or larger. Reversal by washing of edrophon-ium-induced relaxation of rat tracheal rings is shown in Figure 4. Neostigmine at a concentration of 100 µM stimulated rat tracheal ring contraction, and this contraction was attenuated by edrophonium at a concentration of 100 µM. This attenuation was reversed by washing. The effects of edrophonium on neostigmine-induced IP1 accumulation of rat tracheal slices are shown in Figure 5. IP1 accumulation was stimulated by neostigmine at a concentration of 100 µM, and this stimulation was attenuated by edrophonium at a concentration of 100 µM. Neostigmine-induced IP1 accumulation was attenuated by edrophonium at a concentration of 100 µM, and this attenuation was reversed by washing (Fig. 6). Concentration-effect relationships for edrophonium are shown in Figure 7. Decreases in IP1 accumulation at large concentrations were consistent with relaxation of rat tracheal rings.
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Figure 3. Effects of edrophonium on 100 µM neostigmine-induced contraction of rat trachea (mean ± SE; n = 12). ***P < 0.001 versus edrophonium 0 µM.
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Figure 4. Effects of washing on edrophonium-induced relaxation of rat trachea (mean ± SE; n = 6). ***P < 0.001 versus 100 µM edrophonium plus 100 µM neostigmine.
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Figure 5. Effects of edrophonium on 100 µM neostigmine-induced inositol monophosphate (IP1) accumulation of rat trachea (mean ± SE; n = 6–8). *P < 0.05 versus edrophonium 0 µM.
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Figure 6. Effects of washing on edrophonium-induced attenuation of inositol monophosphate (IP1) accumulation of rat trachea (mean ± SE; n = 6). **P < 0.01 versus 100 µM edrophonium plus 100 µM neostigmine.
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Figure 7. Relationship between edrophonium-induced attenuation of inositol monophosphate (IP1) accumulation and edrophonium-induced relaxation in the presence of neostigmine.
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Discussion
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Our results show that edrophonium at large concentrations completely attenuates neostigmine-induced contractile and PI responses of rat trachea. Possible mechanisms involved in the attenuation by edrophonium of neostigmine-induced contraction are as follows. Muscarinic receptors in the airway are divided into M2 and M3 receptors (5,6). The muscarinic M3 receptors exist on airway smooth muscle cell membrane, and there are muscarinic M2 receptors on postganglionic nerve terminals. Stimulation of M3 receptors induces bronchoconstriction, whereas stimulation of M2 receptors inhibits acetylcholine (ACh) release, resulting in attenuation of vagally induced bronchoconstriction. Tanito et al. (7) examined, by a radioligand-binding study, the effects of edrophonium on specific [3H]N-methylscopolamine binding to guinea pig atrial (muscarinic M2 receptors) and submandibular gland (muscarinic M3 receptors) membrane preparations and found that edrophonium inhibited [3H]N-methylscopolamine binding to M2 and M3 receptors in a concentration-dependent manner. They concluded that edrophonium binds to muscarinic M2 and M3 receptors nonselectively and that it acts as a competitive antagonist. In our results, the contraction by neostigmine of rat tracheal rings was completely inhibited by edrophonium. Thus, edrophonium would inhibit muscarinic M3 receptors of airway smooth muscle cell membranes, resulting in the attenuation by edrophonium of neostigmine-induced contraction.
The muscarinic receptor has an orthosteric binding site and an allosteric binding site. The activation of the allosteric binding site changes the association, dissociation, and equilibrium binding of agonists, and it affects the function of the orthosteric binding site (8–11). Endou et al. (12), using isolated, spontaneously beating guinea pig right atrial preparations, reported that neostigmine has two binding sites on the cardiac muscarinic M2 receptor and that the binding site with a high affinity (orthosteric site) for neostigmine would mediate a negative chronotropic effect of the drug. This binding is too tight to be dissociated by washing. However, the binding site with a low affinity (allosteric site) would mediate the antagonistic effect of neostigmine, and the binding is easily dissociated (12). In our results, neostigmine-induced contraction was not easily attenuated, even by repeated washing. Thus, it is likely that small concentrations of neostigmine would also bind tightly to airway muscarinic M3 receptors, as well as cardiac muscarinic M2 receptors. However, it is not clear whether edrophonium has dual effects. We examined the effects of edrophonium on neostigmine-induced contraction of rat trachea because neostigmine was not easily dissociated by repeated washing. Our previous results showed that edrophonium did not affect the resting tension and basal IP1 accumulation of rat trachea (1–3). In these results, large concentrations of edrophonium completely attenuated neostigmine-induced contraction and IP1 accumulation. However, this attenuation was nearly reversed by washing with fresh K-H solution. Yost and Maestrone (13) showed that large concentrations of neostigmine and edrophonium resulted in concentration-dependent inhibition of nicotinic ACh receptors. Thus, edrophonium at large concentrations would bind to the allosteric site, which inhibits the activation by neostigmine of the orthosteric site of muscarinic M3 receptors, resulting in the attenuation of neostigmine-induced contraction through the inhibition of PI response.
Edrophonium binds to muscarinic M3 receptors nonselectively and acts as a competitive antagonist (7). The attenuation by edrophonium of neostigmine-induced contraction was reversed to more than 80% but not to 100% of control levels by washing with fresh K-H solution. Thus, it is likely that edrophonium as a competitive antagonist binds in part to the orthosteric site, resulting in the attenuation of neostigmine-induced contraction.
Other possible mechanisms, such as acetylcholinesterase binding to presynaptic M2 receptors and effects on postganglionic nicotinic receptors, may be considered. These mechanisms are involved in metabolism of ACh, ACh release, or inhibition of ACh release from parasympathetic nerve terminals. First, in the previous study, edrophonium at both smaller and larger concentrations did not affect the resting tension or basal PI response of rat trachea (1–3). The lack of effect by edrophonium suggests that there is no spontaneous ACh release. Second, the attenuation of contraction by neostigmine at large concentrations is not affected by methoctramine, a muscarinic M2 receptor antagonist (1). Third, dimethyl phenylpiperazinium (DMPP), a selective ganglionic nicotinic agonist, does not cause contraction (3). The preparation does not appear to contain a sufficient number of functional postganglionic cells that can be activated by the nicotinic agonists. Thus, it is unlikely that acetylcholinesterase binding to presynaptic M2 receptors and the effects on postganglionic nicotinic receptors would be involved in the attenuation by edrophonium of neostigmine-induced contraction.
When large concentrations of edrophonium are used to reverse the excessive application of nondepolarizing muscle relaxants, edrophonium would bind to the allosteric site of muscarinic M3 receptors, resulting in the inhibition of the orthosteric site and subsequent inhibition of airway contraction. Inhibition of airway contraction may also be seen in patients with myasthenia gravis who have received excessive edrophonium therapy.
In conclusion, edrophonium at large concentrations attenuates neostigmine-induced contractile and PI responses of rat trachea. This attenuation is reversed nearly to control levels by washing. These results suggest that edrophonium would bind to the allosteric site, resulting in the inhibition of the action of the orthosteric site of muscarinic M3 receptors of rat trachea.
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Acknowledgments
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Supported in part by Grant 10671421 for Scientific Research from the Ministry of Education, Science and Culture, Japan.
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Footnotes
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Presented at the 77th annual meeting of the International Anesthesia Research Society, New Orleans, LA, March, 2003.
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References
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Accepted for publication April 30, 2003.
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Abstract
Introduction
