Otenzepad shows two populations of binding sites in human gastric smooth muscle

Cholinergic agonists and antagonists frequently used for gastrointestinal motility disorders often produce adverse effects. A possible explanation for this is the presence of similar muscarinic receptor subtypes on smooth muscle from different gastrointestinal organs. The aim of this study was to characterize muscarinic receptor subtypes in human gastric smooth muscle with receptor binding methods. N-[3H]Methylscopolamine ([3H]NMS) saturation experiments showed a homogeneous population of noninteracting binding sites (KD = 0.76 ± 0.07 nM, Bmax = 46.94 ± 3.69 fmol/mg of tissue protein, nH = 0.99 ± 0.01). The rank order of inhibition of [3H]NMS binding by nonlabelled compounds was atropine [Formula: see text] otenzepad [Formula: see text] pirenzepine. Atropine and pirenzepine bound to a homogeneous population of binding sites. The inhibition of [3H]NMS binding by otenzepad showed two populations of receptors (nH < 1, p < 0.01), whose apparent Ki1 of 298 ± 40 nM and apparent Ki2 of 3.463 ± 0.62 mM were similar to those reported for the M2 and M3 muscarinic receptor subtypes. The M2 subtype was the more abundant of the two, representing 79.12 ± 5.48% of the total population. We conclude that two muscarinic receptor subpopulations similar to the M2 and M3 subtypes are present in human gastric smooth muscle and that the M2-like receptor is the more abundant of the two.Key words: human stomach, muscarinic receptor subtypes, smooth muscle.

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Characterization of muscarinic receptor subtypes in pig airways: radioligand binding and northern blotting studies

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1994.266.6.l642 ◽

1994 ◽

Vol 266 (6) ◽

pp. L642-L648 ◽

Cited By ~ 3

Author(s):

E. B. Haddad ◽

J. C. Mak ◽

A. Hislop ◽

S. G. Haworth ◽

P. J. Barnes

Keyword(s):

Smooth Muscle ◽

Muscarinic Receptor ◽

Airway Smooth Muscle ◽

Binding Sites ◽

Radioligand Binding ◽

Receptor Subtypes ◽

Muscarinic Receptor Subtypes ◽

Selective Antagonist ◽

High Affinity ◽

Peripheral Lung

This study was undertaken to characterize the muscarinic receptor subtypes present in adult pig peripheral lung and airway smooth muscle. The binding of the nonselective muscarinic antagonist [N-methyl-3H]scopolamine ([3H]NMS) to pig airways showed a single class of binding sites with a maximum density of 172 and 450 fmol/mg protein in peripheral lung and airway smooth muscle, respectively. Unlike [3H]NMS, the M1-selective antagonist, [3H]telenzepine, recognized two populations of binding sites in peripheral lung. Approximately 14% of total [3H]telenzepine binding sites displayed high affinity [dissociation constant (Kd) = 0.95 nM], whereas the remaining sites showed low affinity (Kd = 14.2 nM). The high- and the low-affinity [3H]telenzepine binding sites displayed the pharmacological profile of M1 and M2 receptors, respectively. Heterogeneity of pig airways muscarinic receptor was also revealed by competitive binding experiments against [3H]NMS with the M2-selective antagonist methoctramine. This compound recognized 70 and 90% of total receptors with high affinity in airway smooth muscle (Ki = 4.44 nM) and peripheral lung (Ki = 9.82 nM), respectively. This result suggests that the dominant muscarinic receptor in pig airways is of the M2 subtype. Northern blot analysis demonstrated the presence of m1 and m2 mRNAs transcripts in peripheral lung and m2 and m3 mRNAs in airway smooth muscle with no evidence for m4 mRNA.

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Location and quantification of muscarinic receptor subtypes in rat pons: implications for REM sleep generation

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1997.273.3.r896 ◽

1997 ◽

Vol 273 (3) ◽

pp. R896-R904 ◽

Cited By ~ 5

Author(s):

H. A. Baghdoyan

Keyword(s):

Muscarinic Receptor ◽

Reticular Formation ◽

Muscarinic Receptors ◽

Rem Sleep ◽

Binding Sites ◽

Reticular Nucleus ◽

Homogeneous Distribution ◽

Receptor Subtypes ◽

Pontine Reticular Formation ◽

Muscarinic Receptor Subtypes

Microinjecting cholinomimetics into the pontine reticular formation produces a state that resembles natural rapid eye movement (REM) sleep. Evocation of this REM sleeplike states is anatomically site dependent within the pons and is mediated by muscarinic receptors. The cellular and molecular mechanisms underlying cholinergic REM sleep generation and muscarinic receptor subtype involvement remain to be specified. This study tested the hypothesis that muscarinic receptor subtypes are differentially distributed within the oral and caudal divisions of rat pontine reticular nucleus. In vitro receptor autoradiography was used to localize and quantify M1, M2, and M3 binding sites in the pontine reticular formation and in pontine brain stem regions known to regulate REM sleep. M1-M3 binding sites were present in some REM sleep-related nuclei, such as dorsal raphe and locus ceruleus. The pontine reticular formation was found to have a homogeneous distribution of M2 binding sites across its rostral to caudal extent, indicating that anatomic specificity of cholinergic REM sleep induction cannot be accounted for by a differential density of muscarinic receptors.

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Human esophageal smooth muscle cells express muscarinic receptor subtypes M1 through M5

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.2000.279.5.g1059 ◽

2000 ◽

Vol 279 (5) ◽

pp. G1059-G1069 ◽

Cited By ~ 28

Author(s):

Jian Wang ◽

Pawel S. Krysiak ◽

Lisanne G. Laurier ◽

Stephen M. Sims ◽

Harold G. Preiksaitis

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Muscarinic Receptor ◽

Muscle Cells ◽

Receptor Subtypes ◽

Muscarinic Receptor Subtypes ◽

Functional Responses ◽

Selective Antagonist ◽

Esophageal Physiology ◽

Esophageal Smooth Muscle Cells

Receptor characterization in human esophageal smooth muscle is limited by tissue availability. We used human esophageal smooth muscle cells in culture to examine the expression and function of muscarinic receptors. Primary cultures were established using cells isolated by enzymatic digestion of longitudinal muscle (LM) and circular muscle (CM) obtained from patients undergoing esophagectomy for cancer. Cultured cells grew to confluence after 10–14 days in medium containing 10% fetal bovine serum and stained positively for anti-smooth muscle specific α-actin. mRNA encoding muscarinic receptor subtypes M1–M5 was identified by RT-PCR. The expression of corresponding protein for all five subtypes was confirmed by immunoblotting and immunocytochemistry. Functional responses were assessed by measuring free intracellular Ca2+ concentration ([Ca2+]i) using fura 2 fluorescence. Basal [Ca2+]i, which was 135 ± 22 nM, increased transiently to 543 ± 29 nM in response to 10 μM ACh in CM cells ( n = 8). This response was decreased <95% by 0.01 μM 4-diphenylacetoxy- N-methylpiperidine, a M1/M3-selective antagonist, whereas 0.1 μM methoctramine, a M2/M4-selective antagonist, and 0.1 μM pirenzepine, a M1-selective antagonist, had more modest effects. LM and CM cells showed similar results. We conclude that human smooth muscle cells in primary culture express five muscarinic receptor subtypes and respond to ACh with a rise in [Ca2+]i mediated primarily by the M3 receptor and involving release of Ca2+ from intracellular stores. This culture model provides a useful tool for further study of esophageal physiology.

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