Ion transport in a human lens epithelial cell line exposed to hyposmotic and apoptotic stress

• Published in: Journal of cellular physiology 2010-04, Vol.223 (1), p.110-122
• Main Authors: Chimote, Ameet A., Adragna, Norma C., Lauf, Peter K.
• Format: Article
• Language: English
• Subjects: 4-Aminopyridine - pharmacology; Annexin A5 - metabolism; Apoptosis - drug effects; Blotting, Western; Cell Line; Cell Size; Epithelial Cells - drug effects; Epithelial Cells - metabolism; Epithelial Cells - pathology; Humans; Hypotonic Solutions; Immunohistochemistry; Intermediate-Conductance Calcium-Activated Potassium Channels - drug effects; Intermediate-Conductance Calcium-Activated Potassium Channels - genetics; Intermediate-Conductance Calcium-Activated Potassium Channels - metabolism; Ion Transport; Lens, Crystalline - drug effects; Lens, Crystalline - metabolism; Lens, Crystalline - pathology; Osmotic Pressure; Potassium - metabolism; Potassium Channel Blockers - pharmacology; Protein Kinase Inhibitors - pharmacology; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - metabolism; Rubidium - metabolism; Signal Transduction; Staurosporine - pharmacology; Stress, Physiological; Time Factors; Water - metabolism
• ISSN: 0021-9541; 1097-4652
• DOI: 10.1002/jcp.22015

Membrane transport changes in human lens epithelial (HLE‐B3) cells under hyposmotic and apoptotic stress were compared. Cell potassium content, Ki, uptake of the K congener rubidium, Rbi, and water content were measured after hyposmotic stress induced by hypotonicity, and apoptotic stress by the protein‐kinase inhibitor staurosporine (STP). Cell water increased in hyposmotic (150 mOsm) as compared to isosmotic (300 mOsm) balanced salt solution (BSS) by >2‐fold at 5 min and decreased within 15 min to baseline values accompanied by a 40% Ki loss commensurate with cell swelling and subsequent cell shrinkage likely due to regulatory volume decrease (RVD). Loss of Ki, and accompanying water, and Rbi uptake in hyposmotic BSS were prevented by clotrimazole (CTZ) suggesting water shifts associated with K and Rb flux via intermediate conductance K (IK) channels, also detected at the mRNA and protein level. In contrast, 2 h after 2 µM STP exposure, the cells lost ∼40% water and ∼60% Ki, respectively, consistent with apoptotic volume decrease (AVD). Indeed, water and Ki loss was at least fivefold greater after hyposmotic than after apoptotic stress. High extracellular K and 2 mM 4‐aminopyridine (4‐AP) but not CTZ significantly reduced apoptosis. Annexin labeling phosphatidylserine (PS) at 15 min suggested loss of lipid asymmetry. Quantitative PCR revealed significant IK channel expression during prolonged hyposmotic stress. Results suggest in HLE‐B3 cells, IK channels likely partook in and were down regulated after RVD, whereas pro‐apoptotic STP‐activation of 4‐AP‐sensitive voltage‐gated K channels preceded or accompanied PS externalization before subsequent apoptosis. J. Cell. Physiol. 223: 110–122, 2010. © 2009 Wiley‐Liss, Inc.