Journal
JOURNAL OF BIOMOLECULAR SCREENING
Volume 11, Issue 3, Pages 286-295Publisher
SAGE PUBLICATIONS INC
DOI: 10.1177/1087057105285284
Keywords
Curosurf; fusion assay; lamellar bodies; secretion; surfactant
Funding
- Austrian Science Fund FWF [P 17501] Funding Source: Medline
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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 Ca2+ levels nor the ATP-induced Ca2+ 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.
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