Mechanotransduction in A549 Alveolar Cells via Cell Stretch-Induced ATP Release

Abstract

Mechanotransduction at the cellular/tissue level often involves release of signaling molecules. Among them, purines appear to be the most primitive and widespread chemical messengers in the animal and plant kingdoms. Their release is highly mechano-sensitive, but the release pathways and regulatory mechanisms are not well understood. Here, we investigated the effect of unidirectional stretch on ATP release from human lung A549 alveolar cells grown on a flexible substrate. We used real-time luciferin-luciferase bioluminescence imaging combined with IR imaging to simultaneously monitor cellular ATP release and extend of cell stretch. Single 1-s stretch of 15-30% induced transient ATP release that ceased in 2-3 min and was restricted to a limited number of cells. The number of responding cells increased dose-dependently with the extent of stretch but did not involve cell damage. Calibration of the ATP response showed that local ATP concentration in the close proximity (≤150 μm) to stretch-activated cells may exceed 1 μM or even 10 μM. These concentrations are sufficient for autocrine/paracrine stimulation of cell surface purinergic receptors on the neighboring cells. ATP responses were insensitive to putative ATP channel blockers carbenoxolone or NPPB (100 μM), inhibitors of pannexin or anion channels respectively, but were abolished by N-ethylmaleimide. Fluo8 fluorescence measurement of stretch-induced intracellular Ca2+ responses revealed that limited number of cells displayed rapid responses, which peaked in <1-s and ceased in 1-3 min. This is similar to stretch-induced ATP responses and suggests functional connection between the two signals. Experiments show that cell stretch induces ATP release via cell-regulated process, likely exocytosis. Mechano-sensitive ATP release, via autocrine/paracrine effects, initiates purinergic signaling cascade in other cells and may function as a general intercellular mechanotransduction paradigm in the lung and other tissues.