Ouabain-induced cell volume changes and cytotoxicity in LN229 cells
. Oubain-induced cell volume changes and toxicity were inspected in LN229 cell cultures to delineate the time- and concentration-dependent consequences of blocking Na+/K+-ATPase. A. Phase contrast images showing morphological changes of LN229 cells during 6 to 24 hrs exposures to ouabain (1 μM). B. Cell volume distributions examined using a Millipore ScepterTM Handheld Automated cell counter illustrated a dynamic cell volume regulation during 24-hr exposure to ouabain (1 μM). A transient but noticeable cell swelling was seen at 3 and 6 hrs after ouabain treatment, while the cell volume returned to the original size after 12 to 12 hrs in ouabain. C. Cell volume changes were assessed and compared between ouabain (1 uM) in normal medium containing 5 mM K+ and ouabain exposure in an elevated K+ medium (25 mM KCl). Although cells exposed to ouabain in normal medium returned to their original sizes after 24-hr exposure, the ouabain exposure in the high K+ medium eliminated the cell shrinking phase. D. The time- and concentration-dependent cytotoxic effects of ouabain in LN229 cultures. Cell viability was measured using the MTT assay. In general, the longer the exposure time, the lower cell viability was induced by ouabain. Increasing ouabain concentration from 0.1 μM to 1 μM induced further reduction in cell viability. Even higher ouabain concentrations (2 and 5 μM) showed no further increase in toxic effect. Three independent experiments and each group in an experiment contained 3–4 duplicates.