In CHO cells expressing human M1–M4 receptors, 10 μM N-methylscopolamine bromide suppressed calcium elevations persisting after xanomeline exposure and washout. Calcium signals rose again after antagonist washout at M1/M4, but not M2/M3 during the displayed interval. M2/M4 calcium responses used engineered G16 coupling; this result describes antagonism in an agonist-challenge assay. [2]
Equilibrium competition binding at human M3 receptors
Biochemical assay · Membranes from CHO cells stably expressing human M3 muscarinic acetylcholine receptors [1]
Unlabeled NMS displaced [3H]-NMS in an equilibrium competition assay. This is receptor binding affinity, not a functional IC50 or treatment dose.
Literature-Reported Treatment Conditions
- Treatment concentration
- Unlabeled antagonist concentration series; approximately 350 pM [3H]-NMS tracer; 10 μg membrane protein/well
- Exposure duration
- 18–20 h at room temperature
- Method
- Equilibrium radioligand competition; 1.5 ml assay volume; gentle agitation
- Vehicle
- Assay buffer: 10 mM HEPES, 1 mM MgCl2, pH 7.4
- Co-treatment
- [3H]-NMS tracer; nonspecific binding defined with 1 μM atropine
- Reported limits
- Exact NMS concentration series and compound stock solvent are not specified in the retrieved antagonist-Ki method paragraph; no lot stereochemical characterization supplied.
Methods and source locations
Compound reported in the paper: Methscopolamine bromide; Compound label in the paper: N-methylscopolamine (NMS)
Named non-radiolabeled N-methylscopolamine bromide; no isotope equivalence or salt-dose conversion assumed.
Methods: Chemicals and reagents; common binding procedures; Determination of antagonist Ki; Results Table 1 and footnote
Equilibrium affinity at human M3: Mean±SEM; n=10 experiments; Ki derived from displacement IC50 by Cheng–Prusoff correction. — Table 1, N-methyl scopolamine row and footnote
Competition curve slope: Mean±SEM; n=10 experiments. — Table 1, N-methyl scopolamine row and footnote
Competition-derived association and dissociation kinetics at human M3
Biochemical assay · Membranes from CHO cells stably expressing human M3 muscarinic acetylcholine receptors [1]
A competition time-course model estimated association and dissociation rates for unlabeled NMS. Rates depend on this membrane assay and fitting assumptions; they do not establish in vivo residence time or clinical duration.
Literature-Reported Treatment Conditions
- Treatment concentration
- Unlabeled NMS at 10-, 30- and 100-fold its Ki; approximately 1 nM [3H]-NMS; 10 μg membranes/well
- Exposure duration
- Time course; Figure 4D displays observations through approximately 300 min
- Method
- Simultaneous addition of competitor and tracer; 500 μl; room temperature with gentle agitation
- Vehicle
- 10 mM HEPES, 1 mM MgCl2, pH 7.4 assay buffer
- Co-treatment
- [3H]-NMS tracer; 0-competitor time course and 1 μM atropine NSB control
- Reported limits
- Compound stock solvent is not stated in the selected methods. No exact timepoint values inferred from curves.
Methods and source locations
Compound reported in the paper: Methscopolamine bromide; Compound label in the paper: N-methylscopolamine (NMS)
Named non-radiolabeled N-methylscopolamine bromide; no isotope equivalence or salt-dose conversion assumed.
Methods: competition kinetics between [3H]-NMS and unlabelled ligands; Data analysis; Results Figure 4D and caption; Table 2
Modeled association rate of unlabeled NMS: Mean±SEM over ≥4 independent experiments; each concentration time course in quadruplicate; individual-experiment concentration estimates averaged first. — Table 2, N-methyl scopolamine row; competition kinetics Results and Data analysis
Modeled dissociation rate of unlabeled NMS: Same experiment set and SEM convention as kon. — Table 2, N-methyl scopolamine row; competition kinetics Results and Data analysis
Kinetically derived dissociation constant: Derived from koff/kon; table values, not graph estimates; mean±SEM convention. — Table 2, N-methyl scopolamine row
Reversible suppression of xanomeline-associated calcium signals
Cell-based study · CHO cells stably expressing individual human M1–M4 muscarinic receptors; M2/M4 cells additionally transfected with human G16 [2]
After xanomeline exposure and washout, 10 μM NMS returned elevated calcium signals toward basal levels at M1–M4. Washing out NMS restored elevations at M1/M4, but not M2/M3 in the displayed observation interval. This is an agonist-challenge experiment in engineered cells.
Literature-Reported Treatment Conditions
- Treatment concentration
- 10 μM NMS; 10 μM xanomeline for preceding challenge; 300 nM carbachol internal control
- Exposure duration
- Carbachol 5 s, then 5-min washing; xanomeline 20 s, 2-min washing; NMS 2 min, then 4-min buffer washing
- Method
- Fura-2 microfluorometry; normalized 340/380-nm fluorescence ratio; room temperature air-conditioned to 27°C
- Vehicle
- Krebs–HEPES buffer (KHB); drugs diluted directly in KHB unless stated otherwise
- Co-treatment
- NMS applied after xanomeline exposure and washout; carbachol stimulation served as within-cell response control
- Reported limits
- No NMS concentration-response curve or functional IC50 in this experiment. SI Table S6 numerical response parameters were not read or adopted.
Methods and source locations
Compound reported in the paper: Methscopolamine bromide; Compound label in the paper: N-methylscopolamine (NMS)
Named non-radiolabeled N-methylscopolamine bromide; no isotope equivalence or salt-dose conversion assumed.
Methods: Chemicals, Cell culture, transient transfection, Fast microfluorometry; Results: Effects of blockade of the receptor orthosteric binding site on calcium level elevated by xanomeline; Figure 4 and caption
Calcium response during and after NMS application: Representative traces averaged 8–12 best responding cells from one experiment; caption reports confirmation in 5 additional independent experiments. Cell selection limits population representativeness; no exact amplitudes estimated. — Figure 4 image/caption and adjacent Results paragraph
