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

Pflugers Arch. 2016 Nov;468(11-12):2075-2085. doi: 10.1007/s00424-016-1896-4. Epub 2016 Oct 31.

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.

Keywords: Calcium signaling; Osmotic shock; Regulatory volume decrease; VRAC.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Action Potentials
  • Calcium / metabolism*
  • Calcium Channels / metabolism
  • Calcium Signaling
  • Cell Line, Tumor
  • Cell Size*
  • Epithelial Cells / cytology
  • Epithelial Cells / metabolism*
  • Epithelial Cells / physiology
  • Humans
  • Osmotic Pressure*

Substances

  • Calcium Channels
  • Calcium