Transient cholinergic glycoconjugate secretion from swine tracheal submucosal gland cells

By inference, muscarinic stimulation of glycoconjugate release from tracheal submucosal gland cells appears to be a transient, nonequilibrium process (J. M. Farley and T. M. Dwyer, Life Sci. 48: 59-67, 1991). To directly characterize the release kinetics of glycoconjugate, we developed an enzyme-linked lectin assay (ELLA) of much improved precision and resolution. To collect secreted products with an improved time resolution, freshly isolated swine tracheal submucosal gland cells were continuously superfused with medium 199 at 37 degrees, buffered with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) and CO2 or bicarbonate; fractions were collected every 15 s to 2 min. A 30-s pulse of 30 nM acetylcholine (ACh) increased the rate of glycoconjugate release by 10- to 25-fold for 2-3 min. The peak response averaged 14.2 +/- 9.0 ng protein.ml-1.min-1. 4 million cells-1 or 3.6 +/- 2.3 fg.cell-1.min-1 for 30 nM ACh and 16.2 +/- 3.0 ng protein.ml-1.min-1 for 100 nM ACh. There was no significant glycoconjugate release following a 30-s pulse of either 10 nM or 1 microM ACh. A second pulse after 7 min had no measurable effect on glycoconjugate release but a full response was obtained after 30 min. A continuous superfusion begun 1 min following the 30-s pulse resulted in a greater release of glycoconjugate than the pulse alone, but the response was not sustained, falling to twice basal levels within 5 min. We conclude that a brief muscarinic stimulation causes a triggered release of mucus glycoprotein followed by a relative refractory period.