Characterization of muscarinic receptors in dog tracheal smooth muscle cells.

1. The tritiated muscarinic antagonist N-methyl scopolamine, [3H]-NMS, was used to characterize the muscarinic receptors associated with the intact dog tracheal smooth muscle cells. Based on receptor binding assays, the intact tracheal smooth muscle cells had specific, saturable, high-affinity binding sites for [3H]-NMS. 2. Specific binding was cell concentration- and time-dependent. The specific binding of [3H]-NMS was increased linearly with increasing cell concentrations. The equilibrium for association of [3H]-NMS with the muscarinic receptors was attained within 30 min at 37 degrees C. 3. Binding was saturable with respect to [3H]-NMS concentrations. Analysis of binding isotherms yielded an apparent equilibrium dissociation constant (KD) of 320 +/- 20 pM and a maximum receptor density (Bmax) of 13.7 +/- 1.4 fmole per 5 x 10(4) cells. The Hill coefficient for [3H]-NMS binding was 1.00 +/- 0.01. The association (K1) and dissociation (K-1) rate constants were determined to be (1.19 +/- 0.23) x 10(8) M-1 min-1 and 0.034 +/- 0.09 min-1, respectively. KD, calculated from the ratio of K-1 and K1, was 286 +/- 65 pM; this value is close to the value of KD calculated from Scatchard plots of binding isotherms. 4. The non-selective muscarinic antagonist atropine and M1 selective antagonist pirenzepine did not reveal any selectivity of these muscarinic receptors. Pirenzepine competed with [3H]-NMS for a single binding site with a Ki value of (6.02 +/- 0.69) x 10(-7) M which is close to the value of M2 or M3 receptors, indicating that the M1 receptor subtype did not exist in the intact tracheal smooth muscle cells. 5. Competition with cardioselective antagonist (M2), methoctramine; smooth muscle selective antagonists (M3), hexahydrodifenidol and hexahydrosiladifenidol; as well as carbachol, were best fit by a two-binding site model. The results suggest that both M2 and M3 receptor subtypes exist at the cell surface of tracheal smooth muscle cells.