AJP - Cell Physiology, Vol 258, Issue 4 C730-C738, Copyright © 1990 by American Physiological Society
Ligand binding and G protein coupling of muscarinic receptors in airway smooth muscle
P. A. Lucchesi, C. R. Scheid, F. D. Romano, M. E. Kargacin, D. Mullikin-Kilpatrick, H. Yamaguchi and T. W. Honeyman
Department of Physiology, University of Massachusetts Medical School, Worcester 10655.
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 of the muscarinic agonist oxotremorine were
best described as a sum of binding to high- and low-affinity sites with KD
values of 3.8 nM and 2.2 microM. 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 protein
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.(ABSTRACT TRUNCATED AT
250 WORDS)
