Anesth Analg 2004;98:1325-1329
© 2004 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000111104.33104.51
ANESTHETIC PHARMACOLOGY
Metoclopramide Causes Airway Smooth Muscle Relaxation Through Inhibition of Muscarinic M3 Receptor in the Rat Trachea
Masataka Saito, MD*,
Osamu Shibata, MD*,
Masakazu Yamaguchi, MD*,
Maki Yoshimura, MD*,
Tetsuji Makita, MD*,
Noboru Harada, MD*,
Masami Niwa, MD
, and
Koji Sumikawa, MD*
Departments of *Anesthesiology and
Pharmacology 1, Nagasaki University School of Medicine, Nagasaki, Japan
Address correspondence and reprint requests to Masataka Saito, MD, Department of Anesthesia, Kitakyushu-city Yahata Hospital, 4-18-1 Nishihonmachi, Yahatahigashi, Kitakyushu 805-0061, Japan. Address e-mail to saito{at}yahatahp.jp
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Abstract
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Although metoclopramide, often used as an antiemetic, is reported to have an anticholinesterase action, the effect on airway smooth muscle remains unclear. We investigated the effect of metoclopramide on the contraction, phosphatidylinositol response, and binding affinity of muscarinic M3 receptors in rat trachea preparations. Male Wistar rats were anesthetized and their tracheas excised and chopped into 3-mm-wide rings, 1-mm-wide slices, or frozen 10-µm-thick sections. Contraction was induced with 0.55 µM carbachol (CCh) and, 30 min later, metoclopramide (10 µM to 1 mM) was added. The slices were incubated with 3[H]myo-inositol, 0.55 µM CCh, and metoclopramide, and the formation of 3[H] inositol monophosphate was measured. A radioligand binding study was conducted to examine the effects of metoclopramide using [3H] 4-diphenylacetoxy-N-methyl-piperidine methobromide (4-DAMP), a muscarinic M3 receptor antagonist, in sections of the trachea. Metoclopramide concentration dependently attenuated CCh-induced contraction and inositol monophosphate accumulation, and also attenuated the binding affinity of 4-DAMP to muscarinic M3 receptors. The 50% inhibitory concentration of metoclopramide against the binding affinity of 4-DAMP to muscarinic M3 receptors of rat trachea was 24 µM. These findings suggest that the attenuation by metoclopramide of CCh-induced contraction and phosphatidylinositol response may be mediated through the muscarinic M3 receptors.
IMPLICATIONS: We investigated the effect of metoclopramide on the contraction, phosphatidylinositol response, and binding affinity of muscarinic M3 receptors in rat trachea preparations. Our findings suggest that the attenuation by metoclopramide of carbachol-induced contraction and phosphatidylinositol response may be mediated through the muscarinic M3 receptors.
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Introduction
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Postoperative nausea and vomiting are unpleasant for patients and can delay discharge from the postanesthesia care unit or from the hospital in patients undergoing ambulatory surgery. Metoclopramide, often used to treat postoperative nausea and vomiting (1), is a procainamide derivative that has an anticholinesterase effect (2). It is also a dopamine antagonist that binds to D2 receptors in the central and peripheral nervous system. Dopamine affects vagal nerve terminals, and inhibits acetylcholine (ACh) release. MacLaren and Shields (3) reported a case of respiratory failure after oral administration of metoclopramide, and suggested that metoclopramide caused bronchoconstriction, possibly through enhanced vagal nerve activity. Although metoclopramide was reported to have an anticholinesterase action, its effect on airway smooth muscle remains unclear. We investigated the effect of metoclopramide on carbachol (CCh)-induced contractile and phosphatidylinositol (PI) responses, and on the binding affinity of muscarinic M3 receptors in rat trachea preparations.
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Methods
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The studies were conducted under guidelines approved by the Animal Care Committee of Nagasaki University. Twenty-two 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 intraperitoneal), and their tracheas were rapidly isolated.
Contractile Response
Each trachea was cut into 3-mm-wide ring segments with a McIlwain tissue chopper (The Mickle Laboratory Engineering, Gomshall, UK). 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 solution (millimolar 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) and changes in isometric force were recorded using a MacLab system (Milford, MA). The resting tension was adjusted periodically to 1.5 g during the equilibration period. The rings were washed every 15 min and reequilibrated to baseline tension for 60 min (Time 0). The effect of metoclopramide on CCh-induced contractions of rat tracheal rings was examined. At Time 0, CCh, 0.55 µM (80% of the concentration causing maximal contraction of the rat tracheal rings), was added and 30 min later ring relaxation was induced by stepwise cumulative additions of metoclopramide from 10 to 1000 µM in final concentrations.
PI Response
Inositol 1,4,5-trisphosphate (IP3) is rapidly degraded into inositol monophosphate (IP1) and subsequently recycled back to PI via free inositol. Lithium inhibits the conversion of IP1 to inositol. In the presence of Li+, the accumulation rate of IP1 reflects the extent of a PI response. We measured [3H]IP1 in tracheal slices incubated with [3H]myo-inositol (Amersham, Tokyo, Japan) (4–6). The trachea was cut longitudinally and chopped into 1-mm-wide slices with a McIlwain tissue chopper. Three pieces of the tracheal slice were placed in small flat-bottomed tubes and preincubated for 15 min in Krebs-Henseleit solution containing 5 mM LiCl. The solution was continuously aerated with 95% O2/5% CO2. An aliquot of 0.5 µCi [3H]myo-inositol was then added to each tube (a final concentration 0.1 µM in 300 µL incubation volume) and the tubes were flushed with 95% O2/5% CO2, capped, set in a shaking bath at 37°C, and then incubated for 30 min (Time 0). The effect of metoclopramide on CCh-induced IP1 accumulation of rat tracheal slices was examined. At Time 0, varying doses (0–1000 µM) of metoclopramide were added and, 15 min later, 0.55 µM CCh in a final concentration was added. The tubes were reaerated with 95% O2/5% CO2, recapped, and reincubated. After an additional 60-min incubation, 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 the formate form. 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.
Radioligand Binding
A radioligand binding study was conducted to examine the effect of metoclopramide on [3H] 4-diphenylacetoxy-N-methyl-piperidine methobromide (4-DAMP) (7), a muscarinic M3 receptor antagonist of rat trachea. Frozen 10-µm-thick sections of the trachea were cut on a cryostat at –20°C, and thaw-mounted onto gelatin-coated slides. The slides were incubated in buffer with [3H] 4-DAMP (10 nM) and metoclopramide was added. After incubation, the slides were washed and tissue sections were dried under a stream of cold air. Quantitation of radioligand binding was made using the high sensitivity of the computerized radioluminographic system with imaging plates. The values for photostimulated luminescence were obtained directly from the imaging plates by the computerized scanning system. Radioligands binding data were analyzed using the program GraphPad Prism (GraphPad Software, San Diego, CA).
Values were expressed as means ± SE. The results were analyzed by one-way analysis of variance, followed when indicated by Scheffé test. A value of P < 0.05 was considered statistically significant.
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Results
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Figure 1 shows a typical recording of the effect of metoclopramide on CCh-induced contraction of rat tracheal rings. Metoclopramide attenuated CCh-induced contraction of rat tracheal rings. The 50% inhibitory concentration of metoclopramide on CCh-induced tracheal ring contraction was 52 µM (Fig. 2). Metoclopramide attenuated CCh-induced IP1 accumulation in a concentration-dependent manner (Fig. 3). The relation between IP1 formed and the tracheal ring tension is shown in Figure 4. The attenuation by metoclopramide of CCh-induced IP1 accumulation was correlated with relaxation of rat tracheal rings (R2 = 0.646, P < 0.001). Metoclopramide concentration-dependently attenuated the binding affinity of 4-DAMP to muscarinic M3 receptors of rat trachea (Fig. 5). The 50% inhibitory concentration of metoclopramide against the binding affinity of 4-DAMP was 24 µM. The slope factor of this dose response curve was –1.036, which indicated that metoclopramide bound competitively at the muscarinic M3 receptor.
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Figure 1. A typical recording of the effects of metoclopramide (Met) on carbachol (CCh)-induced contraction of rat tracheal rings.
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Figure 2. The effect of metoclopramide on carbachol-induced contraction of rat tracheal rings (mean ± SE; n = 9).
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Figure 3. The effect of metoclopramide on carbachol (CCh)-induced inositol monophosphate (IP1) accumulation of rat tracheal slices (mean ± SE; n = 7–10). CCh = 0.55 µM, Bq = becquerel. ***P < 0.001 versus metoclopramide 0.
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Figure 4. The relation between inositol monophosphate (IP1) formation and the tension of rat tracheal rings. The attenuation by metoclopramide of carbachol (CCh)-induced IP1 formation was correlated with relaxation of rat tracheal rings (R2 = 0.646, P < 0.001). CCh = 0.55 µM, Bq = becquerel.
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Figure 5. The effect of metoclopramide on the specific binding of [3H] 4-diphenylacetoxy-N-methyl-piperidine methobromide (4-DAMP), a muscarinic M3 receptor antagonist in rat tracheal rings (mean ± SE; n = 7–10). The 50% inhibitory concentration of metoclopramide against the binding affinity of 4-DAMP to muscarinic M3 receptors of rat trachea was 24 µM.
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Discussion
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The present results show that metoclopramide attenuated CCh-induced contractile and PI responses of the rat trachea, that the attenuation by metoclopramide of CCh-induced IP1 accumulation was correlated with relaxation of rat tracheal rings, and that metoclopramide also concentration-dependently attenuated the binding affinity of 4-DAMP to muscarinic M3 receptors of the rat trachea.
The possible mechanisms involved in the attenuation by metoclopramide of CCh-induced contraction are as follows: First, metoclopramide may affect muscarinic ACh receptors of airway smooth muscle, resulting in the attenuation of CCh-induced contraction of the rat trachea. The muscarinic M3 receptor exists on airway smooth muscle cell membrane. When the muscarinic M3 receptor in the airway smooth muscle cell membrane is stimulated to activate phospholipase C, IP3 is increased. IP3 mobilizes Ca2+ from sarcoplasmic reticulum, and at the same time Ca2+ influxes from the extracellular space, resulting in an increase in the intracellular Ca2+([Ca2+]i) concentration, in [Ca2+]i which induces bronchoconstriction. In the present study, metoclopramide competitively attenuated the binding affinity of 4-DAMP, a specific antagonist of the muscarinic M3 receptor, and metoclopramide attenuated CCh-induced contractile and PI responses of the rat trachea. Thus, metoclopramide would antagonize the muscarinic M3 receptor of airway smooth muscle competitively, resulting in the attenuation of phospholipase C-coupled PI response, and a subsequent decrease in CCh-induced contraction of rat trachea.
Second, metoclopramide may affect CCh-induced contraction via prejunctional 5-hydroxytryptamine (5-HT) receptors or dopamine D2 receptors of vagal nerve terminals in rat trachea. Takahashi et al. (8) reported that in guinea pig trachea and human bronchi, 5-HT facilitated electric field stimulation-induced cholinergic bronchoconstriction in a concentration-dependent manner, and that 5-HT showed no effect on the contractile response to exogenous ACh, suggesting that 5-HT facilitates cholinergic bronchoconstriction via prejunctional receptors. They observed the effect of 5-HT3 antagonists and selective agonists in human and guinea pig airways, and suggested that these facilitatory effects were mediated by 5-HT3 receptors. Metoclopramide is an antagonist at 5-HT1A and 5-HT3 receptors (9,10). Thus, metoclopramide would inhibit spontaneous ACh release, resulting in attenuation of cholinergic bronchoconstriction. However, dopamine affects vagal nerve terminals in the gastrointestinal tract, and inhibits ACh release. Metoclopramide is also a dopamine antagonist that binds to D2 receptors in the central and peripheral nervous system (11). It inhibits dopamine D2 receptors in the gastrointestinal tract, resulting in increased ACh release from vagal nerve terminals, and a subsequent increase in peristaltic movements.
Conversely, Cabezas et al. (12) examined the possible modulating effect of dopamine on bronchial diameter by administering inhaled dopamine and metoclopramide to subjects with various degrees of bronchial tone and found that metoclopramide did not induce changes in respiratory variables in healthy individuals or in those with asthma without acute bronchospasm. The authors concluded that dopamine D2 blockade with metoclopramide did not modify resting bronchial tone. In the present study, metoclopramide inhibited CCh-induced contraction, but did not affect the resting tension of the rat trachea. It was shown that 1,1-dimethyl-4-phenylpiperazinium iodide, a selective ganglionic nicotinic agonist, does not cause contraction of rat trachea (5), suggesting that isolated tracheal tissue does not contain a sufficient number of functional postganglionic cells and vagal nerve endings. Thus, neither 5-HT receptors nor dopamine D2 receptors would be involved in the attenuation by metoclopramide of CCh-induced contraction of rat trachea.
Third, metoclopramide may enhance cholinesterase activity, resulting in an increase in hydrolysis of CCh and a subsequent attenuation of tracheal contraction. However, Motamed et al. (13) and Skinner et al. (14) reported that metoclopramide induced a slight, but significant, decrease in plasma cholinesterase activity. Chemnitius et al. (15) reported that metoclopramide inhibited acetylcholinesterase isoenzymes in both erythrocytes and human caudate nucleus. Thus, it is unlikely that cholinesterase is involved in the mechanism of action of metoclopramide.
In conclusion, the attenuation by metoclopramide of CCh-induced contractile and PI responses are mediated via the inhibition of muscarinic M3 receptors.
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Acknowledgments
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This study was supported in part by Grant 15591638 for Scientific Research, from the Ministry of Education, Science, and Culture, Japan.
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Footnotes
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Presented at the annual meeting of American Society of Anesthesiologists, San Francisco, CA, October 11–15, 2003.
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References
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Accepted for publication November 19, 2003.
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Abstract
Introduction
