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Cell Height as an Indicator for Volume Changes of Confluent HeLa Cells and the Role of Cellular Ca2+ in Their Regulatory Volume Decrease Response
Chi-Shing WongPo-Yee LuiSamuel KoSiu-Kai Kong
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1999 Volume 7 Issue 3 Pages 113-120

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Abstract

Osmotic cell volume perturbations were approximated by the changes of cell height by scanning HeLa cells loaded with di-8-anepps (a potential-sensitive fluorescent indicator) along the z-axis with a confocal microscope. Cells challenged with a 280 mosM buffer for 10 min did not induce significant changes in cell height. When cells were shocked with a 190 mosM buffer, a rapid cell swelling followed by a slow regulatory volume decrease (RVD) was observed. When the osmolarity of the buffer was adjusted to 170 or 150 mosM, rapid cell swelling was obtained again but cells could not regain their original volume. On the other hand, hypertonic (340 mosM) shock induced cell shrinkage and no regulatory volume increase was observed. Changes in the total area of an optical section 2 μm above the cover slip during 190 mosM hypotonic shock were also determined. Comparison of the changes in cell height to changes in area indicates that cell height was a more sensitive indicator to study cell swelling and RVD in a monolayer of HeLa cells. Results in our study also illustrate that challenge of cells with 190 mosM buffer induced a transient increase in the intracellular free Ca2+ level ([Ca2+]i). When BAPTA/AM-loaded cells were exposed to a Ca2+-free-EGTA 190 mosM buffer, cell swelling was elicited as usual but the rise of [Ca2+]i and the RVD response were abolished. Interestingly, suppression of there-uptake of Ca2+ by thapsigargin (2 μM), a microsomaI Ca2+-ATPase inhibitor, to sustain the increase in [Ca2+]i during the 190 mosM shock did not produce a ‘normal’ but rather a weak and delayed RVD. In contrast, challenge to the cells with the 190 mosM solution in the presence of ionomycin (5 μM) displayed a fast transient Ca2+ response and an accelerated RVD response. These observations therefore indicate that an increase in [Ca2+]i followed by a decrease is an important factor in the triggering of cell volume decrease in HeLa cells.

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