title: Calcium is not required for triggering volume restoration in hypotonically challenged A549 epithelial cells.
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.
authors: Centre de recherche, Centre hospitalier de l'Université de Montréal (CRCHUM), Tour Viger 900 rue St-Denis, Montreal, Quebec, H2X 0A9, Canada.|Faculty of Biology, M.V. Lomonosov Moscow State University, Moscow, Russia.
authors: Olga
authors: O
authors: Ponomarchuk
authors: Centre de recherche, Centre hospitalier de l'Université de Montréal (CRCHUM), Tour Viger 900 rue St-Denis, Montreal, Quebec, H2X 0A9, Canada. francis.boudreault.crchum@gmail.com.
authors: Francis
authors: F
authors: Boudreault
authors: Faculty of Biology, M.V. Lomonosov Moscow State University, Moscow, Russia.
authors: Sergei N
authors: SN
authors: Orlov
authors: Centre de recherche, Centre hospitalier de l'Université de Montréal (CRCHUM), Tour Viger 900 rue St-Denis, Montreal, Quebec, H2X 0A9, Canada. ryszard.grygorczyk@umontreal.ca.|Department of Medicine, Université de Montréal, Montreal, Quebec, Canada. ryszard.grygorczyk@umontreal.ca.
authors: Ryszard
authors: 0000-0002-9798-7651
authors: R
authors: Grygorczyk
mesh_terms: D000200:Action Potentials; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D020013:Calcium Signaling; D045744:Cell Line, Tumor; D048429:Cell Size; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D009997:Osmotic Pressure
chemical_list: D015220:Calcium Channels; D002118:Calcium
keywords: Calcium signaling; Osmotic shock; Regulatory volume decrease; VRAC