Binding characteristics and functional G protein coupling of muscarinic acetylcholine receptors in rat duodenum smooth muscle membranes |
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Authors: | Claus Liebmann Steffen Nawrath Martin Schnittler Harald Schubert Karl-H Jakobs |
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Institution: | (1) Institut für Biochemie and Biophysik, Biologische Fakultät, Friedrich-Schiller-Universität Jena, Philosophenweg 12, 0-6900 Jena, Federal Republic of Germany;(2) Tierexperimentelles Zentrum der Medizinischen Fakultat, Friedrich-Schiller-Universitat Jena, O-6900 Jena, Federal Republic of Germany;(3) Pharmakologisches Institut der Universitat Essen, W-4300 Essen, Federal Republic of Germany |
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Abstract: | Summary The non-selective labelled antagonist 3H]N-methyl-scopolamine (3H]NMS) was used to identify muscarinic acetylcholine receptors in rat duodenum smooth muscle membranes. Saturation and kinetic experiments revealed a binding site with a KD-value of 0.2–0.3 nmol/l and a receptor concentration (Bmax) of 100 fmol/mg protein. The affinities of eight selective muscarinic antagonists were determined and compared with those at M1 (rat cerebral cortex), M2 (rat heart), M3 (rat submandibular gland) and M4 (data from Dörje et al. 1991) receptors. The M2-selective agent AF-DX 116, the group of M2/M4-selective compounds himbacine, AF-DX 384, AQ-RA 741 and methoctramine but also the M3-selective HHSiD showed affinities corresponding to M2 and/or M4 sites. The intermediate affinity of 4-DAMP favours a mixed M2/M4 receptor population mainly containing M2 receptors. Two compounds, pirenzepine and AQ-RA 741, displayed biphasic displacement curves indicating the presence of a small population of putative M1 receptors. The rat duodenum antagonist binding profile, however, is not consistent with the presence of M3 receptors. We further demonstrate a concentration-dependent stimulation of 35S]GTPS] binding to duodenal G proteins by the muscarinic agonist oxotremorine. Estimation of the binding parameters of GTPS] in absence and presence of oxotremorine provided evidence for a catalytic activation of G proteins by agonist-activated muscarinic receptors in rat duodenal membranes and a strong signal amplification on the G protein level.
Send offprint requests to C. Liebmann at the above address |
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Keywords: | Rat duodenum Muscarinic receptors M2/M4/M1 subtypes Catalytic G protein activation |
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