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Calcium is not required for triggering volume restoration in hypotonically challenged A549 epithelial cells

  • Signaling and cell physiology
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Pflügers Archiv - European Journal of Physiology Aims and scope Submit manuscript

Abstract

Maintenance of cell volume is a fundamental housekeeping function in eukaryotic cells. Acute cell swelling activates a regulatory volume decrease (RVD) process with poorly defined volume sensing and intermediate signaling mechanisms. Here, we analyzed the putative role of Ca2+ signaling in RVD in single substrate-adherent human lung epithelial A549 cells. Acute cell swelling was induced by perfusion of the flow-through imaging chamber with 50 % hypotonic solution at a defined fluid turnover rate. Changes in cytosolic Ca2+ concentration ([Ca2+]i) and cell volume were monitored simultaneously with ratiometric Fura-2 fluorescence and 3D reconstruction of stereoscopic single-cell images, respectively. Hypotonic challenge caused a progressive swelling peaking at ∼20 min and followed, during the next 20 min, by RVD of 60 ± 7 % of the peak volume increase. However, at the rate of swelling used in our experiments, these processes were not accompanied by a measurable increment of [Ca2+]i. Loading with intracellular Ca2+ chelator BAPTA slightly delayed peak of swelling but did not prevent RVD in 82 % of cells. Further, electrophysiology whole-cell patch-clamp experiments showed that BAPTA did not block activation of volume-regulated anion channel (VRAC) measured as swelling-induced outwardly rectifying 5-nitro-2-(3-phenylpropyl-amino) benzoic acid sensitive current. Together, our data suggest that intracellular Ca2+-mediated signaling is not essential for VRAC activation and subsequent volume restoration in A549 cells.

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Acknowledgments

This study was supported by Natural Science and Engineering Research Council of Canada discovery grant RGPIN 435517-2013 (RG).

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Authors and Affiliations

  1. Centre de recherche, Centre hospitalier de l’Université de Montréal (CRCHUM), Tour Viger 900 rue St-Denis, Montreal, Quebec, H2X 0A9, Canada

    Olga Ponomarchuk, Francis Boudreault & Ryszard Grygorczyk

  2. Faculty of Biology, M.V. Lomonosov Moscow State University, Moscow, Russia

    Olga Ponomarchuk & Sergei N. Orlov

  3. Department of Medicine, Université de Montréal, Montreal, Quebec, Canada

    Ryszard Grygorczyk

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  1. Olga Ponomarchuk
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  2. Francis Boudreault
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  4. Ryszard Grygorczyk
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Correspondence to Francis Boudreault or Ryszard Grygorczyk.

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Ponomarchuk, O., Boudreault, F., Orlov, S.N. et al. Calcium is not required for triggering volume restoration in hypotonically challenged A549 epithelial cells. Pflugers Arch - Eur J Physiol 468, 2075–2085 (2016). https://doi.org/10.1007/s00424-016-1896-4

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  • DOI: https://doi.org/10.1007/s00424-016-1896-4

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