Involvement of the M2 muscarinic receptor in contractions of the guinea pig trachea, guinea pig esophagus, and rat fundus

doi:10.1016/0006-2952(95)02396-8

Biochemical Pharmacology

Volume 51, Issue 6, 22 March 1996, Pages 779-788

https://doi.org/10.1016/0006-2952(95)02396-8 Get rights and content

Abstract

The involvement of the M₂ muscarinic receptor in contractile responses of the guinea pig trachea, guinea pig esophagus, and rat fundus was investigated. In the standard assay, oxotremorine-M elicited contractions of the trachea with an ec₅₀ value of approximately 73 nM. [[2-[(Diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116) at 1 and 10 μM antagonized these contractions by 2.1- and 9.0-fold increases in the ec₅₀ value for oxotremorine-M. These effects are consistent with antagonism of an M₃-mediated contractile response. In subsequent experiments, the M₃ receptors were first inactivated selectively by incubation with N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard) (40 nM) for 1 hr in the presence of AF-DX 116 (1 μM) followed by extensive washing. In 4-DAMP mustard treated trachea, oxotremorine-M elicited contractions with an ec₅₀ value of 0.31 μM in the presence of histamine (10 μM) and forskolin (4 μM). Under these conditions, AF-DX 116 at 1 and 10 μM antagonized contractions to oxotremorine-M by 8- and 59-fold increases in the ec₅₀, respectively, while para-fluorohexahydrosiladiphenidol (p-F-HHSiD) (0.1 μM) had no effect. These effects are consistent with a contraction being mediated by an M₂ receptor. In the guinea pig esophagus and rat fundus, AF-DX 116 and p-F-HHSiD blocked contractions measured under similar conditions with magnitudes intermediate between what would be expected from an M₂ and an M₃ receptor, suggesting that perhaps both subtypes contribute to the overall contractile response under these conditions. In addition, contractions of the guinea pig trachea measured in the presence of histamine and forskolin were pertussis toxin sensitive. These results indicate that, in the trachea, M₂ receptors can dominate the contractile response after a majority of the M₃ receptors have been inactivated, whereas in the guinea pig esophagus and rat fundus, M₂ receptors may contribute to, but do not play a dominant role in the overall response.

References (51)

AD Michel et al.
Methoctramine reveals heterogeneity of M₂ muscarinic receptors in longitudinal ileal smooth muscle membranes
Eur J Pharmacol
(1988)
FJ Ehlert et al.
Functional role of M₂ muscarinic receptors in the guinea pig ileum
Life Sci
(1995)
A Maeda et al.
Tissue distribution of mRNAs encoding muscarinic acetylcholine receptor subtypes
FEBS Lett
(1988)
AD Fryer et al.
Identification of three muscarinic receptor subtypes in rat lung using binding studies with selective antagonists
Life Sci
(1990)
TJ Torphy
Differential relaxant effects of isoproterenol on methacholine versus leukotriene D₄-induced contraction in the guinea-pig trachea
Eur J Pharmacol
(1984)
N Watson et al.
Role of muscarinic M₂ and M₃ receptors in guinea-pig trachea: Effects of receptor alkylation
Eur J Pharmacol
(1995)
Y Kamikawa et al.
Heterogeneity of muscarinic receptors in the guinea pig esophageal muscularis mucosae and ileal longitudinal muscle
Gastroenterology
(1985)
E Monferini et al.
Characterization of the muscarinic receptor in the rat urinary bladder
Eur J Pharmacol
(1988)
K Kashihara et al.
Cloning of the rat M3, M4 and M5 muscarinic acetylcholine receptor genes by the polymerase chain reaction (PCR) and the pharmacological characterization of the expressed genes
Life Sci
(1992)
T Katada et al.
Direct modification of the membrane adenylate cyclase system by islet activating protein due to ADP-ribosylation of a membrane protein
J Biol Chem
(1982)

H Kurose et al.

Specific uncoupling by islet-activating protein, pertussis toxin, of negative signal transduction via α-adrenergic, cholinergic, and opiate receptors in neuroblastoma × glioma hybrid cells

J Biol Chem

(1983)

G Lambrecht et al.

p-Fluoro-hexahydrosila-difenidol: The first M₂β-selective muscarinic antagonist

Eur J Pharmacol

(1988)

AF Roffel et al.

Characterization of the muscarinic receptor subtypes involved in phosphoinositide metabolism in bovine tracheal smooth muscle

Br J Pharmacol

(1990)

LM Candell et al.

Differential coupling of subtypes of the muscarinic receptor to adenylate cyclase and phosphoinositide hydrolysis in the longitudinal muscle of the rat ileum

Mol Pharmacol

(1990)

CM Yang

Characterization of muscarinic receptors in dog tracheal smooth muscle cells

J Auton Pharmacol

(1991)

EA Thomas et al.

A functional role for the M₂ muscarinic receptor in smooth muscle of guinea pig ileum

Mol Pharmacol

(1993)

EA Thomas et al.

Pertussis toxin blocks M₂ muscarinic receptor-mediated effects on contraction and cyclic AMP in the guinea pig ileum, but not M₃-mediated contractions and phosphoinositide hydrolysis

J Pharmacol Exp Ther

(1994)

H Reddy et al.

Characterization of the interaction between muscarinic M₂ receptors and β-adrenoceptor subtypes in guinea-pig isolated ileum

Br J Pharmacol

(1995)

G Lambrecht et al.

Pharmacology of hexahydro-difenidol, hexahydro-sila-difenidol, and related selective muscarinic antagonists

Trends Pharmacol Sci

(1989)

L Noronha-blob et al.

Muscarinic receptors: Relationships among phosphoinositide breakdown, adenylate cyclase inhibition, in vitro detrusor muscle contractions and in vivo cystometrogram studies in guinea pig bladder

J Pharmacol Exp Ther

(1989)

L Zhang et al.

Muscarinic receptors in canine colonic circular smooth muscle. I. Coexistence of M₂ and M₃ subtypes

Mol Pharmacol

(1991)

CM Yang et al.

Muscarinic receptor subtypes coupled to generation of different second messengers in isolated tracheal smooth muscle cells

Br J Pharmacol

(1991)

M Del Tacca et al.

Differential affinities of AF-DX 116, atropine and pirenzepine for muscarinic receptors in guinea pig gastric fundus, atria and urinary bladder: Might atropine distinguish among muscarinic receptor subtypes?

Pharmacology

(1990)

Z Li et al.

Functional and localization of high and low affinity binding sites to muscarinic receptors in longitudinal and circular smooth muscles of human stomach

Res Commun Chem Pathol Pharmacol

(1990)

RM Eglen et al.

Comparison of the muscarinic receptors of the guinea-pig oesophageal muscularis mucosae and trachea in vitro

J Auton Pharmacol

(1988)

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In addition, activation of muscarinic M2 receptor contributes to, but do not play a dominant role in SMD-induced contractions. It has been clarified that contractions induced by agonist of muscarinic receptor are not mainly determined by muscarinic M2 receptor (Thomas and Ehlert, 1996). Muscarinic receptor, including muscarinic M1–5 receptors, are widely distributed in stomach smooth muscle (So et al., 2003).
Simo Decoction (SMD), a traditional Chinese medicine, included four elements, such as Fructus aurantii, Radix aucklandiae, Semen arecae and Radix linderae. It has been used to improve gastrointestinal dysmotility in clinical practice for a long history in China. However, the explicit mechanisms are unclear. The aim of this study was to investigate the effect of SMD on contractions of antral circular smooth muscle strips of Sprague-Dawley (SD) rats and its underlying mechanism.
The antral circular strips were prepared in the organ bath under baseline or to be incubated with muscarinic receptor antagonist atropine (10⁻⁶ M), muscarinic M3 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) (0.4×10⁻⁶ M), muscarinic M2 receptor antagonist gallamine (10⁻⁶ M), adrenergic receptor agonist adrenaline (10⁻⁷ M), exogenous nitric oxide (NO) donor l-arginine (10⁻⁴ M), nicotinic receptor antagonist hexamethonium chloride (10⁻⁴ M) and Ca²⁺ channel antagonist nifedipine (30 nM), and consecutive concentrations of SMD were added to the bath to observe the strip responses. As a control, the responses of strips after administration with the same volume of Krebs solution as SMD were also noted. The strip responses to acetylcholine (10⁻⁷–10⁻³ M) were also noted in organ bath to compare with SMD-induced contraction.
SMD dose-dependently evoked hypercontractility of antral circular strips, and the maximal contractile effect of circular smooth muscle induced by SMD was significantly higher than that induced by acetylcholine (10⁻³ M). The responses of antral circular strips to SMD were completely antagonized by atropine, 4-DAMP or 4-DAMP+gallamine, but partly inhibited by gallamine and partly suppressed by adrenaline, l-arginine, hexamethonium chloride and nifedipine.
SMD promotes contractions of antral circular strips in rats mainly via activation of muscarinic M3 receptor, but partly via activation of muscarinic M2 receptor, Ca²⁺ channel and nicotinic receptor, inhibition of adrenergic receptor and releasing of NO.
In vitro effect of bethanechol and suberyldicholine on regions of guinea pig esophagus
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Citation Excerpt :
Despite the anatomical differences, muscle cells of the guinea pig and human have been shown to express similar agonist receptors. For example, the muscarinic receptor M3 has been demonstrated as the primary mediator of contractions in both guinea pig and human smooth muscle esophageal cells [14, 15]. The functional assessment of tissue engineered esophageal constructs requires both an understanding of the contractile parameters of native esophagus tissue and a consistent methodology for the stimulation and measurement of muscular contractions.
Tissue engineering and regenerative medicine is envisaged as the future option for esophageal replacement; however, engineering of a functional esophagus is impeded by the limited understanding of the anatomical complexity of this dynamic muscular organ. The aim of this study was to characterize the function of native esophageal tissue and determine differences in functional response to stimulation between anatomical sites.
The in-vitro response of guinea pig esophageal preparations, from various anatomical sites, to muscle agonists was investigated. Esophageal strips were exposed to bethanechol, an agonist of muscarinic receptors located on smooth muscle, and suberyldicholine, an agonist of nicotinic receptors located on striated muscle, within a Schuler organ bath, to determine the contractile response on the various segments of the esophagus.
The esophagus responded in a reliable and consistent manner to agonist stimulation. Bethanechol dose response curves were constructed with doses of 10 to 300 μM. The average maximal contractions to bethanechol exposure were 4.51, 4.80, 5.55, and 9.15 mN for upper, upper middle, lower middle, and lower esophageal segments, respectively. Responses to singular stimulation with 30 μM suberyldicholine in the presence of tetrodotoxin (100 μM) gave average contractions of 1.07, 0.84, 2.60, and 3.02 mN for upper, upper middle, lower middle, and lower esophageal segments, respectively. Bethanechol and suberyldicholine-induced responses were greater in the lower esophagus in comparison to the upper esophageal segments.
These findings pave the way for the use of an in-vitro bethanechol and suberyldicholine-induced contraction model for future assessment of engineered esophageal tissue.
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The aerial parts of Sisymbrium officinale Scop. are commonly used to treat airway ailments, moreover in antiquity the herbal drug was reputed to possess anticancer properties. The results obtained in present work support the traditional use and the properties ascribed to Sisymbrium officinale.
In order to give a scientific basis to the traditional uses of Sisymbrium officinale, this study was aimed to evaluate in vitro the myorelaxant activity, the antimicrobial properties and the antimutagenic effect of an aqueous dry extract of the aerial parts of the plant. A phytochemical characterization of the extract was also performed.
The myorelaxant activity was studied against the contractions induced by carbachol, histamine and leukotriene C₄, in isolated guinea-pig trachea. The antimicrobial activity was tested against six bacteria and one yeast. The Ames test, performed by the preincubation method, was used to study the antimutagenic activity of the extract by its capability to inhibit the mutagenic effect of 2-nitrofluorene, sodium azide, methyl methanesulfonate and 2-aminoanthracene, in Salmonella typhimurium TA98, Salmonella typhimurium TA100 and Escherichia coli WP2uvrA strains. The chemical composition of the extract was analyzed by TLC and HPLC.
Sisymbrium officinale showed to reduce the chemically-induced contractions of isolated guinea-pig trachea with major potency against leukotriene C₄ and histamine. The extract did not show any antibacterial activity. The Ames test showed a strong antimutagenic activity against 2-aminoanthracene, in Escherichia coli WP2uvrA and in Salmonella typhimurium TA98 strains. The phytochemical study highlighted the presence of putranjivine, the glucosinolate marker of Sisymbrium officinale, and of proline.
The myorelaxant activity of Sisymbrium officinale offers a scientific basis to its use in traditional medicine. The strong antimutagenic effect suggests further studies to evaluate its possible chemopreventive activity.
Contractile role of M<inf>2</inf> and M<inf>3</inf> muscarinic receptors in gastrointestinal, airway and urinary bladder smooth muscle
2003, Life Sciences
Both M₂ and M₃ muscarinic receptors are expressed in smooth muscle and influence contraction through distinct signaling pathways. M₃ receptors interact with G_q to trigger phosphoinositide hydrolysis, Ca²⁺ mobilization and a direct contractile response. In contrast, M₂ receptors interact with G_i and G_o to inhibit adenylyl cyclase and Ca²⁺-activated K⁺ channels and to potentiate a Ca²⁺-dependent, nonselective cation conductance. Ultimately, these mechanisms lead to the prediction that the influence of the M₂ receptor on contraction should be conditional upon mobilization of Ca²⁺ by another receptor such as the M₃. Mathematical modeling studies of these mechanisms show that the competitive antagonism of a muscarinic response mediated through activation of both M₂ and M₃ receptors should resemble the profile of the directly acting receptor (i.e., the M₃) and not that of the conditionally acting receptor (i.e., the M₂). Using a combination of pharmacological and genetic approaches, we have identified two mechanisms for the M₂ receptor in contraction: 1) a high potency inhibition of the relaxation elicited by agents that increase cytosolic cAMP and 2) a low potency potentiation of contractions elicited by the M₃ receptor. The latter mechanism may be involved in muscarinic agonist-mediated heterologous desensitization of smooth muscle, which requires activation of both M₂ and M₃ receptors.
The mechanisms of α<inf>2</inf>-adrenoceptor agonist-induced contraction in longitudinal muscle of the porcine uterus
2000, European Journal of Pharmacology
The aim of the present study was to clarify the cellular mechanisms underlying the α₂-adrenoceptor-mediated contraction of porcine myometrium (nonvascular smooth muscle). Acetylcholine (3 nM–1 μM), clonidine (1 nM–10 μM) and 5-bromo-N-[2-imidazolin-2-yl]-6-quinoxalinamine (UK14304) (1 nM–10 μM) in Krebs solution caused a concentration-dependent contraction in the longitudinal muscles of the porcine uterus with similar EC₅₀ values and maximum responses. A lowered external Ca²⁺ concentration and verapamil (10 nM–10 μM) decreased the contractile response to clonidine and UK14304 more markedly than the response to acetylcholine. However, in Kumagai solution, neither clonidine nor UK14304 caused contractile responses, but acetylcholine remained effective. The effects of α₂-adrenoceptor agonists on intracellular Ca²⁺ concentration ([Ca²⁺]_i) and smooth muscle force were measured simultaneously using fura-PE3-loaded muscle preparations. Clonidine and UK14304 caused increases in [Ca²⁺]_i and force of the longitudinal muscle. The increases in [Ca²⁺]_i and muscle force were markedly inhibited by verapamil and in Ca²⁺-free solution (EGTA, 1 mM). In the absence of external Ca²⁺, clonidine caused only a small increase in [Ca²⁺]_i in Ca²⁺-loaded preparations compared with those increases caused by carbachol, histamine, and oxytocin. Ca²⁺ (2.5 mM) caused increases in [Ca²⁺]_i and force of the longitudinal muscles in a Ca²⁺-free high K⁺ solution. Clonidine concentration dependently potentiated the Ca²⁺-induced contraction without significantly changing the increase in [Ca²⁺]_i, and this potentiation was inhibited by yohimbine. These results suggested that clonidine increases the Ca²⁺ sensitivity of the contractile elements through activation of α₂-adrenoceptors. During the development of the contractile response to clonidine (1 μM, 0–5 min), tissue cyclic AMP levels did not change significantly. In vitro treatment with pertussis toxin (1 μg/ml for 2 h) significantly decreased the contraction induced by clonidine without affecting the responses to carbachol and high K⁺. The present results indicate that in porcine myometrium, α₂-adrenoceptor stimulation caused contraction of the longitudinal muscles by mechanisms largely dependent on the influx of extracellular Ca²⁺, probably through voltage-dependent Ca²⁺ channels (VDCCs), and that the potentiation of the Ca²⁺ sensitivity of the contractile elements is another mechanism of the contractile responses. These actions involve a pertussis-toxin-sensitive G protein (probably G_i type) in the signal transduction pathway.
Subtypes of muscarinic receptors in rat duodenum: A comparison with rabbit vas deferens, rat atria, guinea-pig ileum and gallbladder by using imperialine
1999, General Pharmacology
The specific binding of [³H]QNB to rat duodenum smooth muscle membranes was a saturable process and Scatchard transformation of the saturation curves indicated a linear plot $n_{H} = 1.017 ± 0.071$ . The K_D and B_max values were 0.168 ± 0.025 nM and 46.7 ± 8.6 fmol/mg protein, respectively. Analyses of competition curves using pirenzepine and guanylpirenzepine indicated more than one class of binding site. A minor population of muscarinic binding sites showed high affinity (M₁) for both pirenzepine $19.3 ± 1.2%; pK_{i} = 8.29 ± 0.36$ and guanylpirenzepine $29.4 ± 2.0%; pK_{i} = 7.28 ± 0.11$ . The antagonistic affinity values of pirenzepine and guanylpirenzepine for the remaining low affinity binding sites, and that of methoctramine indicated the presence of both M₂ and M₃ subtypes. McN-A-343 produced relaxations in rat duodenum and inhibited twitch contractions of rabbit vas deferens induced by electrical stimulation in a concentration dependent manner. Carbachol (Cch) exerted concentration-dependent negative inotropic effect in rat atria and contractile effects in guinea-pig gallbladder and ileum longitudinal muscle-myenteric plexus preparation. Imperaline displaced the concentration-response curves to McN-A-343 and Cch to the right in parallel, without affecting the maximum responses in all tissues studied. The rank order of the pA₂ values was rabbit vas deferens > rat atria > guinea-pig gallbladder = guinea-pig ileum > rat duodenum. The presynaptic muscarinic receptors at the rat duodenum and rabbit vas deferens were concluded to be of M₁ and M₄ subtypes, respectively.

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^☆: This work was supported by N.I.H. Grants NS 26511 and NS 30882.

^∗: Recipient of an Advanced Predoctoral Fellowship from the Pharmaceutical Manufacturers Association Foundation. Present address: Scripps Research Institute, Department of Molecular Biology, M.B. 10, 10666 North Torrey Pines Rd., La Jolla, CA 92037.

^†: Recipient of a United States Public Health Service Research Career Development Award (NS01396) from the National Institute of Neurological Disorders and Stroke.

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Research paperInvolvement of the M2 muscarinic receptor in contractions of the guinea pig trachea, guinea pig esophagus, and rat fundus☆

Abstract

Eur J Pharmacol

Life Sci

FEBS Lett

Life Sci

Eur J Pharmacol

Eur J Pharmacol

Gastroenterology

Eur J Pharmacol

Life Sci

J Biol Chem

J Biol Chem

Eur J Pharmacol

Characterization of the muscarinic receptor subtypes involved in phosphoinositide metabolism in bovine tracheal smooth muscle

Br J Pharmacol

Differential coupling of subtypes of the muscarinic receptor to adenylate cyclase and phosphoinositide hydrolysis in the longitudinal muscle of the rat ileum

Mol Pharmacol

Characterization of muscarinic receptors in dog tracheal smooth muscle cells

J Auton Pharmacol

A functional role for the M2 muscarinic receptor in smooth muscle of guinea pig ileum

Mol Pharmacol

Pertussis toxin blocks M2 muscarinic receptor-mediated effects on contraction and cyclic AMP in the guinea pig ileum, but not M3-mediated contractions and phosphoinositide hydrolysis

J Pharmacol Exp Ther

Characterization of the interaction between muscarinic M2 receptors and β-adrenoceptor subtypes in guinea-pig isolated ileum

Br J Pharmacol

Pharmacology of hexahydro-difenidol, hexahydro-sila-difenidol, and related selective muscarinic antagonists

Trends Pharmacol Sci

Muscarinic receptors: Relationships among phosphoinositide breakdown, adenylate cyclase inhibition, in vitro detrusor muscle contractions and in vivo cystometrogram studies in guinea pig bladder

J Pharmacol Exp Ther

Muscarinic receptors in canine colonic circular smooth muscle. I. Coexistence of M2 and M3 subtypes

Mol Pharmacol

Muscarinic receptor subtypes coupled to generation of different second messengers in isolated tracheal smooth muscle cells

Br J Pharmacol

Differential affinities of AF-DX 116, atropine and pirenzepine for muscarinic receptors in guinea pig gastric fundus, atria and urinary bladder: Might atropine distinguish among muscarinic receptor subtypes?

Pharmacology

Functional and localization of high and low affinity binding sites to muscarinic receptors in longitudinal and circular smooth muscles of human stomach

Res Commun Chem Pathol Pharmacol

Comparison of the muscarinic receptors of the guinea-pig oesophageal muscularis mucosae and trachea in vitro

J Auton Pharmacol

Research paper
Involvement of the M₂ muscarinic receptor in contractions of the guinea pig trachea, guinea pig esophagus, and rat fundus☆

A functional role for the M₂ muscarinic receptor in smooth muscle of guinea pig ileum

Pertussis toxin blocks M₂ muscarinic receptor-mediated effects on contraction and cyclic AMP in the guinea pig ileum, but not M₃-mediated contractions and phosphoinositide hydrolysis

Characterization of the interaction between muscarinic M₂ receptors and β-adrenoceptor subtypes in guinea-pig isolated ileum

Muscarinic receptors in canine colonic circular smooth muscle. I. Coexistence of M₂ and M₃ subtypes