Provocholine is inhaled using a standardized procedure. The challenge is performed by giving a subject ascending serial concentrations of Provocholine. At each concentration, five breaths are administered by a nebulizer that permits intermittent delivery time of 0.6 seconds by a breath-actuated timing device (dosimeter). At each of five inhalations of a serial concentration, the subject begins at functional residual capacity (FRC) and slowly and completely inhales the dose delivered. Within 5 minutes, FEV1 values are determined. The procedure ends either when there is a 20% or greater reduction in the FEV1 compared with the baseline sodium chloride solution value (i.e., a positive response) or if 188.88 total cumulative units have been administered and the FEV1 has been reduced by 14 % or less (i.e., a negative response). If there is a reduction of 15% to 19% in the FEV, compared with baseline, either the challenge may be repeated at that concentration or a higher concentration may be given as long as the dosage administered does not result in total cumulative units exceeding 188.88.

Agonist activity of methacholine was determined using assay of accumulation of [3H]inositol monophosphate. The CHO cells were labeled in 12-well plates with 1 uCi/mL myo-[3H]inositol for 24 h. Wells contained ca. 3* 10^5 cells in a volume of 1 mL. Cells were then washed once with Hank’s balanced salt media containing 20 mM HEPES buffer (pH 7.4) and were then incubated in the buffered Hank’s media containing 10 mM LiCl at room temperature for 15 min. Muscarinic agents were then added to each well. After incubation for 1 h at 37 °C, the medium was aspirated and replaced with 750 uL of cold 20 mM aqueous formic acid. After 35 min at 4 °C, the formic acid was removed and replaced with 250 uL of 60 mM NH4OH. The inositol monophosphate in the NH4OH solution was isolated by anionexchange chromatography19 and counted for tritium in Hydrofluor scintillation fluid. Cells were treated with 0.001 - 10 uM of methacholine, EC50 was determined to be 120 nM.