A fluorescent microplate assay for exocytosis in alveolar type II cells.
The authors describe a simple, reliable, and quantitative assay to monitor exocytotic fusion of lamellar bodies (LBs) in adherent rat alveolar type II (AT II) cells. The assay is based on fluorescence measurements of LB-plasma membrane (PM) fusions modified for the use in multiwell culture plates to obtain a high-sample throughput. In particular, it is based on the presence of a highly light-absorbing dye in the cell supernatants to increase the specificity of fluorescence signals and to yield pseudo-confocal information from the cells. When the assay was tested with agonist-(ATP) and phorbolester-induced stimulation of LB-PM fusions, the authors found a good correlation with direct microscopic investigations based on single cell recordings. To further validate the assay, they used Curosurf at 10 mg/ml. However, it influenced neither the basal nor the ATP-stimulated rate of LB-PM fusions. This was corroborated by the fact that Curosurf had no effect on resting Ca (2+) levels nor the ATP induced Ca (2+) signals. The results cast new light on previous findings that surfactant phospholipids decrease the rate of secretion in AT II cells in a dose-dependent way. The authors conclude that the inhibitory effect exerted by phospholipids might be due to action on a later step in exocytosis, probably associated with exocytotic fusion pore expansion and content release out of fused vesicles.
Wemhöner
A
A
Department of Pediatrics, Division of Neonatology, Medical University of Innsbruck, Innsbruck, Austria.
Frick
M
M
Dietl
P
P
Jennings
P
P
Haller
T
T
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D005089:Exocytosis; D005453:Fluorescence; D008297:Male; D011650:Pulmonary Alveoli / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000255:Adenosine Triphosphate; D013755:Tetradecanoylphorbol Acetate