Induced sputum was collected the day after measurements of cough reflex sensitivity, spirometry, and cough peak flow were made. Capsaicin-containing meals were not allowed for 12 h before sputum collection. Around 2 h after the intake of their regular dopaminergic medication, the sputum collection was performed. A 3% saline solution was administered via an ultrasonic nebulizer for 15 to 30 min until the sputum volume was approximately 1 mL. Because the sample contained saliva, we eliminated this contamination by visual inspection and inverted microscopy examination.27–
The SP was quantified using modification of a previously described method.28
The collected 1-mL sputum samples were immediately mixed with 0.5 U/mL aprotonin and 3 mmol/L ethylenediaminetetraacetic acid, and was stored at −70°C until assay. For radioimmunoassay, the samples were mixed with 2 vol (vol/vol) acetone using a mixer at room temperature for 5 min. The precipitate was pelleted by centrifugation at 2,500g for 5 min. After centrifugation, the supernatants were extracted twice with petroleum ether, and the supernatant then was evaporated in a water bath under N2 gas. After evaporation, the dried residue was dissolved in a 0.05 mol/L Tris-HCl buffer (pH 8.65) containing 0.1% human serum albumin, 0.01 mmol/L ethylenediaminetetraacetic acid, 0.15 mmol/L NaCl, 0.002% sodium azide, and 0.2 mL samples were incubated with 0.05 mL rabbit anti-SP serum (SRL, Inc; Tokyo, Japan) for 24 h at 4°C. 125I SP, 15,000 counts per minute (NEN Life Science Products, Inc; Boston, MA) in 0.05 mL, was added, and the mixture was incubated for an additional 24 h at 4°C. Bound and free ligands were separated by adding 0.05 mol/L Tris-HCl buffer, 1% γ-globulin, and 25% polyethyleneglycol (6,000 mol/L), and were centrifuged at 1,700g for 20 min. The radioactivity in the precipitate was counted in a γ-spectrometer.