Proceedings of The Physiological Society
University of Manchester (2010) Proc Physiol Soc 19, C32
Ouabain-induced caspase activity but not volume decrease is attenuated by 4, 4-diisothiocyanatostilbene-2, 2-disulfonic acid (DIDS) in Jurkat cells.
L. Morris2, A. Collins1
1. Centre for Vision & Vascular Science, Queen's University, Belfast, Co Antrim, United Kingdom. 2. Centre for Biomedical Education, Queen's University, Belfast, Co Antrim, United Kingdom.
Ouabain is well known both as an inhibitor of the Na+-K+ pump and as an inducer of apoptosis. It has been proposed that these two effects are mechanistically related in that Na+-K+ pump inhibition leads to K+ efflux as a requirement for the initiation of apoptotic signalling pathways (1). On the other hand, the Na+-K+ pump is considered to be essential for the control of cell volume by the well-accepted ‘pump-leak’ mechanism by which Cl- influx and osmotic swelling are prevented by membrane polarisation. Accordingly, inhibition of the Na+-K+ pump would lead to cell swelling and lysis. Despite this, apoptosis is almost always characterised by cell shrinkage, and in at least some cells is accompanied by a decrease in Na+-K+ pump activity even in the absence of ouabain (2). This suggests that apoptotic cells have an alternative pumping mechanism for the control of cell volume. We hypothesised a mechanism in which the metabolic generation of H+ fuels Cl- efflux via the Cl--HCO3- exchanger. Jurkat cells were treated with ouabain and the Cl--HCO3- exchange inhibitor 4, 4-diisothiocyanatostilbene-2, 2-disulfonic acid (DIDS). Apoptosis was assayed by the Caspase-Glo® assay (Promega) and cell size was monitored by analysis of digital brightfield microscopy images (CellProfiler (3)). Cell profile area after 6hr in 100μM ouabain (o) was not consistently different from control (c). In 4 separate experiments, c 145±2 (739), o 144±2 (822); c 126±1 (870), o 115±1 (1094); c 143±1 (1059), o 140±2 (674); c 132±1 (991), o 149±2 (879) [mean±sem μm2 (n)]. 100μM DIDS (d) had little or no effect on cell profile area, either in the presence or absence of ouabain. In the first two of the above experiments, d 145±1 (1147), o+d 146±2 (885) and d 131±1 (1015), o+d 125±2 (705). Caspase 3/7 activity was increased after 6hr by 100μM ouabain (3300±242 (3) relative light units vs 1012±13 (3) for control; p<0.001 by ANOVA) but not by 1.5μM DIDS, although DIDS reduced the effect of ouabain (o+d 2090±116 (3); p<0.01 vs o). Cells had characteristic apoptotic morphology after 19hr in 100μM ouabain. Again 100μM DIDS had little or no effect on cell profile area in the absence and presence of ouabain. In 2 separate experiments, cell profile areas were c 117±1 (1307), o 43±3 (48), d 113±2 (692), o+d 44±3 (110); c 123±2 (760), o 65±3 (183), d 115±2 (763) , o+d 72±3 (195). By ANOVA, c vs o p<0.001 and d vs o+d p<0.001 in both experiments. These results indicate that DIDS can inhibit the pro-apoptotic action of ouabain, and suggest that apoptotic Jurkat cells have a volume control mechanism that does not require the Na+-K+ pump or the Cl--HCO3- exchanger.
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