Imaging and characterization of stretch-induced ATP release from alveolar A549 cells

Authors

  • Ryszard Grygorczyk,

    1. Research Centre, Centre hospitalier de l’Université de Montréal (CRCHUM) – Hôtel-Dieu, and Department of Medicine, Université de Montréal, Montreal, Quebec, Canada
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  • Kishio Furuya,

    1. FIRST Research Center for Innovative Nanobiodevices, Nagoya University, Nagoya, Japan
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  • Masahiro Sokabe

    1. FIRST Research Center for Innovative Nanobiodevices, Nagoya University, Nagoya, Japan
    2. Department of Physiology, Nagoya University, Graduate School of Medicine, Nagoya, Japan
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R. Grygorczyk: CRCHUM − Hôtel-Dieu, 3850 St. Urbain St., Montreal, Quebec, Canada H2W 1T8. Email: ryszard.grygorczyk@umontreal.ca

Key point

  • Translation of mechanical forces into biological effects often involves ATP release and activation of the purinergic signalling cascade.

  • In the lungs, stretch is an important physical stimulus but its impact on ATP release and intercellular signalling is poorly understood.

  • Here, we report that in the A549 alveolar cell model, a single stretch of more than 10% induces ATP release sufficient to stimulate purinergic receptors on neighbouring cells up to 150 μm away from the source cell.

  • Stretch-induced ATP release is significantly enhanced during healing in scratch-wound areas.

  • These results demonstrate that mechano-purinergic signalling may play an important role in modulating alveolar processes, including electrolyte transport, surfactant secretion and wound healing.

Abstract  Mechano-transduction at cellular and tissue levels often involves ATP release and activation of the purinergic signalling cascade. In the lungs, stretch is an important physical stimulus but its impact on ATP release, the underlying release mechanisms and transduction pathways are poorly understood. Here, we investigated the effect of unidirectional stretch on ATP release from human alveolar A549 cells by real-time luciferin–luciferase bioluminescence imaging coupled with simultaneous infrared imaging, to monitor the extent of cell stretch and to identify ATP releasing cells. In subconfluent (<90%) cell cultures, single 1 s stretch (10–40%)-induced transient ATP release from a small fraction (≤1.5%) of cells that grew in number dose-dependently with increasing extent of stretch. ATP concentration in the proximity (≤150 μm) of releasing cells often exceeded 10 μm, sufficient for autocrine/paracrine purinoreceptor stimulation of neighbouring cells. ATP release responses were insensitive to the putative ATP channel blockers carbenoxolone and 5-nitro-2-(3-phenylpropyl-amino) benzoic acid, but were inhibited by N-ethylmaleimide and bafilomycin. In confluent cell cultures, the maximal fraction of responding cells dropped to <0.2%, but was enhanced several-fold in the wound/scratch area after it was repopulated by new cells during the healing process. Fluo8 fluorescence experiments revealed two types of stretch-induced intracellular Ca2+ responses, rapid sustained Ca2+ elevations in a limited number of cells and delayed secondary responses in neighbouring cells, seen as Ca2+ waves whose propagation was consistent with extracellular diffusion of released ATP. Our experiments revealed that a single >10% stretch was sufficient to initiate intercellular purinergic signalling in alveolar cells, which may contribute to the regulation of surfactant secretion and wound healing.

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