Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC61
Stability of Jurkat cell size in ATP-depleting and furosemide-containing media.
E. McGreevy1, A. Collins1
1. Centre for Vision & Vascular Science, Queen's University Belfast, Belfast, United Kingdom.
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. Controlled cell death by apoptosis is accompanied by a decrease in Na+-K+ pump activity in at least some cell types, including Jurkat cells, but cells typically maintain their volume or shrink rather than swell during apoptosis. Ouabain is a Na+-K+ pump inhibitor that induces apoptosis without cell swelling in Jurkat cells, suggesting the possibility of an alternative pumping mechanism for extruding osmolytes. Depletion of ATP would be expected to cause cell swelling if volume control was dependent on an ATP-driven pump. Jurkat cells were incubated in control RPMI medium or in ATP-depleting conditions: 1 μM rotenone; glucose replaced with 2-deoxyglucose (2-DG; 10 mM)1,2. Digital micrographs were taken periodically and analysed for cell profile area using CellProfiler software3. Profile areas (mean ± sem µm2, n) were initially 123 ± 1.0, 1328 (control) and 122 ± 1.6, 769 (2-DG + rotenone) and were hardly changed after 5 hr incubation: 125 ± 1.1, 1312 (control) and 119 ± 0.9, 1569 (2-DG + rotenone). By 9 hr in 2-DG + rotenone the cells had condensed nuclei characteristic of apoptosis. One alternative mechanism for volume stability in the face of Na+-K+ pump inhibition depends on concurrent Cl- efflux down an outward gradient set up by Na+-Cl- cotransport via carriers such as NKCC4. Inhibition of such cotransport would cause cell shrinkage. The profile area of Jurkat cells hardly changed during 8.5 hr incubation with 100 μM furosemide, a NKCC inhibitor (initial control 117 ± 1.9, 502; initial furosemide 127 ± 2.9, 627; 8.5 hr control 123 ± 1.1, 1672; 8.5 hr furosemide 121 ± 1.1, 1454). Cells swelled in hypotonic (122 mosm/kg) RPMI (200 ± 1.4, 1872) and returned to control size within 2.6 hr (134 ± 0.9, 2378). This regulatory volume decrease was prevented by 100 μM ouabain (206 ± 2.0, 1503 at 2.6 hr) despite a lack of effect of 100 μM ouabain on cell size in normotonic RPMI (initial 139 ± 1.1, 1309; 2.6 hr 139 ± 0.8, 2341). These results provide no evidence for ATP-dependent pumps or furosemide-sensitive Cl- uptake in normotonic volume control in Jurkat cells. Experiments reported here were repeated with similar results.
Where applicable, experiments conform with Society ethical requirements