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

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• 作者：Olga Ponomarchuk ; Francis Boudreault…
• 关键词：VRAC ; Osmotic shock ; Regulatory volume decrease ; Calcium signaling
• 刊名：Pflügers Archiv - European Journal of Physiology
• 出版年：2016
• 出版时间：November 2016
• 年：2016
• 卷：468
• 期：11-12
• 页码：2075-2085
• 全文大小：
• 刊物主题：Human Physiology; Molecular Medicine; Neurosciences; Cell Biology; Receptors;
• 出版者：Springer Berlin Heidelberg
• ISSN：1432-2013
• 卷排序：468

文摘

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.