Ligand binding and G protein coupling of muscarinic receptors in airway smooth muscle

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Abstract
Ligand binding properties of muscarinic receptors were examined in membranes and isolated cells prepared from bovine trachea. The binding of the muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) to both membranes and isolated cells was saturable, reversible, and of high affinity [dissociation constant (KD) = 100-200 pM]. The binding constants of three selective antagonists, pirenzepine, AF-DX 116, and 4-DAMP, were examined, and the results indicate that the smooth muscle cells contain at least two receptor subtypes. The majority of receptors exhibit binding constants for these selective antagonists similar to those of the M2-subtype. AF-DX 116 binding curves indicated the presence of a small population of receptors with binding constants similar to those reported for the M3-subtype. These findings suggest that the smooth muscle cells may contain both M2- and M3-receptors and are in agreement with evidence of the presence of mRNAs coding for these two subtypes in tracheal extracts (A. Maeda, T. Kubo, M. Mishina, and S. Numa. FEBS Lett. 239: 339-342 1988). [3H]QNB displacement curves as a sum of binding to high- and low-affinity sites with KD values of 3.8 nM and 2.2 .mu.M. Guanosine 5''-O-(3-thiotriphosphate) (GTP.gamma.S) shifted the high-affinity sites to low affinity, suggesting that the high-affinity sites may represent receptors coupled to G proteins. Pertussis toxin catalyzed the ADP ribosylation of a 40- to 41-kDa band present in the membranes but had no significant effect on high-affinity agonist binding, suggesting that most of the receptors are coupled to G proteins in a toxin-insensitive manner. This diversity of receptor subtypes and G proteins may account for the diversity of cholinergic responses exhibited by airway smooth muscle.

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