Tirf Microscopy Study of Exocytotic ATP Release from A549 Epithelial Cells

Abstract

Extracellular nucleotides, via interaction with cell surface purinergic receptors, regulate multiple physiological processes in the lungs, including airway mucociliary clearance and surfactant secretion. Release of ATP from nonexcitable cells can be provoked by mechanical perturbations and cell-swelling, but the underlying mechanisms remain incompletely understood. We have shown previously that cell-swelling induced ATP secretion from A549 cells tightly correlates with intracellular Ca2+ elevations and sought to establish whether Ca2+-dependent exocytosis is involved. In this study, 50% hypotonic shock-induced ATP release from A549 cells was examined by total internal reflection fluorescence (TIRF) microscopy in an attempt to directly visualize ATP-loaded vesicle movement, recruitment and fusion with the plasma membrane. Cells were loaded with quinacrine, a fluorescent marker of ATP intracellular storage sites, and time-lapse imaging was performed using through the objective TIRF system. The time-course of fluorescence intensity changes of individual quinacrine-stained vesicles was evaluated during 1-2-min following hypoosmotic stimulation. Approximately 20%-30% of vesicles visible by TIRF at the cell base showed a quasi-instantaneous disappearance during the first minute post-stimulation, as expected for vesicle fusion and dispersal of their content. This was accompanied by recruitment of ∼10% new vesicles into the evanescent field followed by their exocytosis. The hypotonic stimulus significantly (∼5-fold) increased rate of exocytotic events compared to rate of spontaneous events in unstimulated cells. Exocytotic release mechanism is also consistent with ATP efflux measurements using luciferin-luciferase luminescence assay. Agents known to disrupt exocytotic process (brefeldin, monensin), or cytoskeleton (nocodazole, cytocholasin) reduced ATP release significantly (by up to 80%), while the release was completely blocked by N-ethylmaleimide (1 mM), and low (10oC) temperature. Thus, hypotonic shock-induced ATP secretion from A549 cells occurs mainly via Ca2+-dependent exocytosis. Supported by CIHR and CCFF (RG), Emerging Technologies Fund Grant Texas (Z.G.), NIH-HL090786 (JB).