Effect of Volume Changes on Ouabain-insensitive Net Outward Cation Movements in Human Red Cells

The effect of cell volume changes in human red cells on ouabain-insensitive net outward cation movements through 1) the Na-K and Li-K cotransport, 2) the Li-Na counter-transport system and 3) the furosemide-insensitive Na, K and Li pathway was studied. Cell volume was altered by changing a) the internal cation content (isosmotic method) or b) the external osmolarity of the medium (osmotic method). Na-K and Li-K cotransport were measured as the furosemide-sensitive Na or Li and K efflux into (Na, Li and K)-free (Mg-sucrose replacement) medium from cells loaded to contain approximately equal concentrations of Na and K, or a constant K/Li concentration ratio of 9:1, respectively. Li-Na countertransport was assayed as the Na-stimulated Li efflux from Li-loaded cells and net furosemide-insensitive outfluxes in (Na, Li and K)-free media containing 1 mM furosemide. Swelling of cells by the isosmotic, but not by the osmotic method reduced furosemide-sensitive Na and Li but not K efflux by 80 and 86%, respectively. Changes in cell volume by both methods had no effect on Li-Na countertransport. The effects of cell volume changes were measured on the rate constants of ouabain- and furosemide-insensitive cation fluxes and were found to be complex. Isosmotic shrinkage more than doubled the rate constants of Na and Li efflux but did not affect that of K efflux. Osmotic shrinkage increased the K efflux rate constant by 50% only in cells loaded for countertransport. Isosmotic cell swelling specifically increased the K+ efflux rate constants both in cells loaded for cotransport and countertransport assays while no effect was observed in cells swollen by the osmotic method. Thus, the three transport pathways responded differently to changes in cell volume, and, furthermore, responses were different depending on the method of changing cell water content.