[3H]QNB binding and contraction of rabbit colonic smooth muscle cells

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.