Effects of xanomeline or carbachol pretreatments on 0.2 nM [3H]NMS binding in CHO hM1, rM1, or mutant123 cells.

Cells were pretreated with increasing concentrations of xanomeline or carbachol for 1 h or 24 h at 37°C followed by washing and immediate use in the binding assay or further incubation in the absence of free xanomeline for 23 h. Cells were then incubated with 0.2 nM [3H]NMS at 37°C for 1 h. Data were corrected for protein as indicated in results. Parameters derived from nonlinear regression analysis are shown as mean ± S.E.M. of four to six experiments conducted in triplicate.aNegative logarithm of the IC50 for binding to a single affinity site.bNegative logarithm of the IC50 for the high-affinity agonist binding site; percentage of binding sites shown in parentheses.cNegative logarithm of the IC50 for the low-affinity agonist binding site.dMaximal percentage inhibition of [3H]NMS binding.eControl, naïve cells were incubated simultaneously with agonist and radioligand.fResults shown for xanomeline in control CHO hM1 cells are from nonlinear regression analysis with the bottom constrained to be greater than 0.gNot measured.*Significant difference (p50 between control and 1 h washout as determined by students unpaired t-test.†Significant difference (p50 between the indicated groups and 1 h washout as determined by one-way ANOVA with Dunnett’s post-test.‡Significant difference (pmax from 100 percent.