Mechanosensetive ATP Release Involves a Non-Conductive Pathway: Evidence from Large Field of View Real-Time Imaging

Abstract

Cell swelling is a potent ATP secretion stimulus. While the sources of ATP are known, it's mechanisms of secretion in epithelial cells remain unclear. Two secretion pathways have been proposed: exocytosis of nucleotide-containing vesicles or conductive release through pore-forming channels, like pannexins or connexins. In this project, we have tested whether ATP is released or not through a conductive pathway in A549 lung epithelial cells. Real-time ATP secretion from glass-adherent A549 cells was imaged with a custom-designed lens system, combining a large field of view and a high light-gathering power, mounted directly on a Photometrics® EvolveTM 512 EMCCD camera. ATP was detected with a light-emitting luciferin-luciferase reaction. Secretion was stimulated by acute cell swelling or exposure to ionomycin (10 µM). Plasma membrane permeability to large molecules was assessed in parallel by propidium iodide (PI) uptake with epifluorescence Zeiss Axio Observer microscope. Cells were grown in 10% (control cells) or 0.1% FBS (fragile cells). Our novel imaging system revealed a strong homogeneous ATP secretion lasting several minutes from control A549 cells stimulated with 50% hypotonic shock. Yet, no PI-stained cells were detected under these conditions. In contrast, cells rendered fragile, and thus prone to cell lysis, showed an appreciable amount of PI-stained cells post-hypotonic shock and a punctuate pattern of ATP secretion co-localizing with PI-positive cells. Sudden intracellular calcium elevation with ionomycin provoked a rapid, albeit small, ATP secretion. Based on the fact that hypotonically-induced ATP release in normal conditions was not accompanied with detectable permeation of PI, we conclude that ATP in A549 cells is released by a non-conductive pathway.