A Fluorescent Microplate Assay for Exocytosis in Alveolar Type II Cells

Abstract

The authors describe a simple, reliable, and quantitative assay tomonitor exocytotic fusion of lamellar bodies (LBs) in adherent rat alveolar type II (AT II) cells. The assay is based on fluorescence measurements of LB-plasmamembrane (PM) fusions modified for the use inmultiwell 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 pseudoconfocal 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. Thiswas corroborated by the fact that Curosurf had no effect on resting Ca ²⁺ levels nor the ATPinduced 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-dependentway. The authors conclude that the inhibitory effect exerted by phospholipidsmight be due to action on a later step in exocytosis, probably associated with exocytotic fusion pore expansion and content release out of fused vesicles.