At 30-min intervals, selleck chem Wortmannin the apical medium was collected and stored. We added experimental drugs prior to the fourth time period and compared the amount of mucin secreted during this period with the previous 30-min period. The ratio of mucin release after drug exposure during the fourth period to release during the third period was determined for each cell sheet and compared with control cell sheets not receiving drugs. Thus secretion is expressed as (P4/P3)test/(P4/P3)control, where P4 and P3 are the amounts of mucus released during the third and fourth collection periods, respectively. For example, suppose in control tissues the amount of mucin released during period 4 was 77% of that released in period 3, whereas in test tissues treated with secretagogue the amount of mucin released in period 4 was 150% of that released in period 3.
Then the stimulated secretory rate was 194% of control (i.e., 150/0.77). Secretagogues tested included methacholine (MCh), phenylephrine (Phe), isoproterenol (Iso), and ATP at concentrations ranging from 10?7 to 10?4 M. These agonists were selected for the following reasons: MCh because acetylcholine is the most potent stimulatory of fluid and mucin secretion by native glands; Phe because it has negligible effects on fluid secretion by human glands; Iso because in native glands it stimulates mucin release but has relatively minor effects on fluid secretion; and ATP because it is an efficacious secretagogue of goblet and gland cells (36, 43, 57, 59). In some studies of non-CF mucous cells, diphenylamine-2-carboxylic acid (DPAC) at 10?3 M was added during the final 30-min period.
We used a sandwich-type ELISA to quantify mucin secretion (61). We coated 96-well microtiter plates (Immulux HB; Dynex Technologies, Chantilly, VA) with 100 ��l of a 5 ��g/ml solution of purified monoclonal antibody A10G5 (17). The plates were incubated overnight (4��C) and then washed five times with PBS. Next, the wells were incubated (2 h, room temperature) with 0.1% gelatin in PBS to block nonspecific binding of antigen. After five washes with PBS containing 0.1% Tween 20 (PT), undiluted experimental samples (100 ��l) were added in triplicate wells and incubated for 1 h at room temperature. After five washes with PT, wells were incubated (1 h, room temperature) with 100 ��l of 10 ��g/ml biotinylated A10G5 (28) diluted in ELISA buffer (3% normal goat serum, 0.
05% Tween GSK-3 20 in PBS). The plates were washed five times in PT and then incubated (1 h, room temperature) with streptavidin-��-galactosidase conjugate (Rockland Immunochemicals, Gilbertsville, PA) (1:1,000 in ELISA buffer). Following five washings with PT, 100 ��l of 2-nitrophenyl-��-d-galactopyranoside, 1 mg/ml in 0.05 M NaPO4, pH 7.2, 1.5 mM MgCl2 was applied to each well. After 30 min, absorbance values were read at 405 nm, via a microplate reader (Model 3550, Bio-Rad, Richmond, CA).
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