AJP - Gastrointestinal and Liver Physiology, Vol 253, Issue 5 656-G661, Copyright © 1987 by American Physiological Society
[3H]QNB binding and contraction of rabbit colonic smooth muscle cells
M. J. Ringer, P. E. Hyman, H. W. Kao, C. T. Hsu, T. Tomomasa and W. J. Snape Jr
Department of Medicine, Harbor-UCLA Medical Center, Torrance 90509.
We used radioligand binding and studies of cell contraction to characterize
muscarinic receptors on dispersed smooth muscle cells from rabbit proximal
and distal colon. Cells obtained after serial incubations in collagenase
were used to measure binding of tritiated quinuclidinyl benzilate [(
3H]QNB). AT 37 degrees C, specific [3H]QNB binding was saturable and
linearly related to cell number. Nonlinear regression analysis was used to
determine the affinity of [3H]QNB for its receptor. In the distal colon the
Kd was 60 pM, and the mean number of receptors was 1.2 X 10(6)/cell.
Compared with cells from the distal colon, cells from the proximal colon
had a lower affinity (Kd = 337 pM) but similar numbers of receptors. The
concentrations of the antagonists atropine, secovorine, and pirenzepine,
which were required for inhibition of 50% [3H]QNB binding (IC50), were 8,
5, and 870 nM, respectively, suggesting that the receptors are of the
M2-muscarinic subclass. Hill coefficients for these agents were 1.1, 0.9,
and 1.1, suggesting binding to a single receptor. The IC50 for the
muscarinic agonists bethanechol and oxotremorine were 80 and 0.57 microM,
respectively. Hill coefficients were 0.67 for both, suggesting more complex
interactions involving receptors of different affinities. In studies of
cell contraction, bethanechol stimulated a dose-dependent decrease in cell
length with half the maximal contraction occurring at 100 pM. These results
suggest that 1) contraction is mediated by binding of bethanechol to
M2-muscarinic receptors and that 2) there are a large number of spare
receptors in colonic smooth muscle.
