Norma Adragna (Committee Member), Thomas Brown (Committee Member), Peter Lauf (Advisor), Daniel Organisciak (Committee Member), Robert Putnam (Committee Member), Panagiotis Tsonis (Committee Member)
Doctor of Philosophy (PhD)
Living cells maintain constant volume in response to physiological stresses by altering trans-membrane ion, solute and water flow. In the present study, early and late membrane transport changes in human lens epithelial (HLE-B3) cells under hyposmotic and apoptotic stress were compared. Cell potassium (Ki), rubidium (Rbi)-uptake and water content were measured by atomic absorption spectrophotometry and gravimetry, respectively. Intracellular chloride concentration [Cl]i was determined with the fluorescence dye N- (ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE), during regulatory volume decrease (RVD) after hyposmotic stress and apoptotic volume decrease (AVD) induced by staurosporine (STP), a protein-kinase inhibitor. Cell water increased in hyposmotic balanced salt solution (BSS) as compared to isosmotic BSS by > 2-fold at 5 min and decreased within 30 min to baseline values, accompanied by K efflux commensurate with cell swelling and RVD. Clotrimazole (CTZ) reduced Ki and water loss in hyposmotic BSS, and Rbi uptake, suggesting a role for intermediate conductance K (IK) channels during RVD. This was verified by reverse transcriptase polymerase chain reaction (RT-PCR), Western blot and immunochemistry of HLE-B3 cells.
Apoptosis measured 2 h after 2 μM STP treatment was significant and accompanied by 20% and 41% water loss after 30 min and 2 h, respectively. During apoptosis, Ki fell at a rate of 0.007/min until 30 min and decreased to 42% by 2 h. [Cl]i decreased by 25% and 19% below baseline values at 30 min and 60 min, respectively. High extracellular K, and 2 mM 4-aminopyridine (4-AP) significantly reduced apoptosis whereas the presence of CTZ did not. Annexin labeling appeared at 15 min suggesting lipid changes preceded the water and K loss initiating AVD. In contrast, loss of K, water, and most likely Cl differed by an order of magnitude between RVD and AVD at early time points. Quantitative PCR revealed decreased expression of IK channels during prolonged hyposmotic and apoptotic stress. These studies suggest that in HLE-B3 cells, IK channels partake in RVD, but cannot yet be discounted in AVD, which in part commences through 4-AP-sensitive voltage-gated K channels
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