title: Ion transport in a human lens epithelial cell line exposed to hyposmotic and apoptotic stress.
abstract: Membrane transport changes in human lens epithelial (HLE-B3) cells under hyposmotic and apoptotic stress were compared. Cell potassium content, K(i), uptake of the K congener rubidium, Rb(i), 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% K(i) loss commensurate with cell swelling and subsequent cell shrinkage likely due to regulatory volume decrease (RVD). Loss of K(i), and accompanying water, and Rb(i) 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 microM STP exposure, the cells lost approximately 40% water and approximately 60% K(i), respectively, consistent with apoptotic volume decrease (AVD). Indeed, water and K(i) 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.
authors.affiliation: Cell Biophysics Group, Boonshoft School of Medicine, Wright State University, Dayton, Ohio 45435, USA.
authors.forename: Ameet A
authors.initials: AA
authors.lastname: Chimote
authors.forename: Norma C
authors.initials: NC
authors.lastname: Adragna
authors.forename: Peter K
authors.initials: PK
authors.lastname: Lauf
mesh_terms: D015761:4-Aminopyridine / Q000494:pharmacology; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002460:Cell Line; D048429:Cell Size; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D007038:Hypotonic Solutions; D007150:Immunohistochemistry; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D017136:Ion Transport; D007908:Lens, Crystalline / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D009997:Osmotic Pressure; D011188:Potassium / Q000378:metabolism*; D026902:Potassium Channel Blockers / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012413:Rubidium / Q000378:metabolism; D015398:Signal Transduction; D019311:Staurosporine / Q000494:pharmacology; D013312:Stress, Physiological; D013997:Time Factors; D014867:Water / Q000378:metabolism
chemical_list: D017304:Annexin A5; D007038:Hypotonic Solutions; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; D026902:Potassium Channel Blockers; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D014867:Water; D015761:4-Aminopyridine; D019311:Staurosporine; D012413:Rubidium; D011188:Potassium