Sodium-Potassium Pump Inhibition and Cell Volume
A suspension of human erythrocytes is placed in isotonic buffer at 37°C. Ouabain is added at 1 mM, fully inhibiting the Na+/K+-ATPase. Within 2 hours, intracellular Na+ rises from 10 mM to 60 mM, K+ falls from 140 mM to 90 mM, and cell volume increases 20%. The buffer contains 140 mM Na+, 5 mM K+, and 150 mM impermeant anions. The cells have a high density of aquaporin-1 and a Na+/K+/2Cl− cotransporter that is inwardly directed. ATP levels fall only 10%, because glycolysis continues. The experimenter then removes extracellular Na+.
1. Explain mechanistically why the cells swell after ouabain, invoking osmotic forces and the role of the pump.
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The Na+/K+-ATPase normally exports 3 Na+ and imports 2 K+ per ATP, creating an electrochemical gradient and, critically, keeping total intracellular solute (and thus osmolarity) below the external value so water is not drawn in. Ouabain blocks this, so Na+ leaks in down its gradient (10 → 60 mM) and K+ leaks out; the inward Na+/K+/2Cl− cotransporter adds further solute. Because the membrane has high water permeability via aquaporin-1, water follows osmotically. Intracellular osmoles rise, water enters, and the cell swells 20%. Although ATP is only 10% lower, ATP supply is not the limiting factor-the pump is pharmacologically inhibited, so glycolytic ATP cannot restore ion gradients. Judgment: swelling is a colloid-osmotic (Donnan) effect unmasked by pump failure, not an energy failure.
2. Predict the effect of removing extracellular Na+ on cell volume and on the Na+/K+/2Cl− cotransporter.
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Removing extracellular Na+ eliminates the inward Na+ gradient, so Na+ leak influx stops and the Na+/K+/2Cl− cotransporter (which requires Na+ on the outside) cannot operate. Net solute entry falls, so osmotic water influx slows or reverses; the cells should shrink toward or below their original volume, though K+ efflux continues and regulatory volume decrease mechanisms (K+ and Cl− channels) may be engaged. Some swelling may persist if intracellular impermeant anions and retained Na+ keep osmolarity high, but the driving force for further swelling is gone. Judgment: volume regulation depends on the Na+ gradient, not just on pump activity; removing external Na+ converts a swelling condition into a shrinking tendency.