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A Fluorescent Microplate Assay for Exocytosis in Alveolar Type II Cells

Department of Pediatrics, Division of Neonatology, Medical University of Innsbruck, Innsbruck, Austria

M. Frick

MRC-Laboratory of Molecular Biology, Cambridge, United Kingdom

P. Dietl

Department of General Physiology, University of Ulm, Ulm, Germany

P. Jennings

Department of Physiology and Medical Physics, Division of Physiology, Medical University of Innsbruck, Innsbruck, Austria

T. Haller

Department of Physiology and Medical Physics, Division of Physiology, Medical University of Innsbruck, Fritz-Pregl-Str. 3, A-6020 Innsbruck, Austria thomas.haller{at}i-med.ac.at

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.

Key Words: Curosurf • fusion assay • lamellar bodies • secretion • surfactant

Journal of Biomolecular Screening, Vol. 11, No. 3, 286-295 (2006)
DOI: 10.1177/1087057105285284


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