Ouabain-uninhibited sodium transport in human erythrocytes. Evidence against a second pump

Others have concluded that a second Na "pump" (active Na outflux) exists in human erythrocytes. This second pump was said to be ouabain-insensitive, unlike the classic ouabain-sensitive Na-K pump. An alternative explanation is that "pump II" is Na exchange diffusion. These hypotheses were examined in the present experiments, utilizing (22)Na influx and outflux measurements, net Na fluxes, and ATPase determinations. Ouabain-uninhibited Na outflux was reduced 0.58+/-0.05 mmol/liter cells per h when extracellular Na (Na(o)) was replaced by Mg. Ethacrynic acid or furosemide produced similar decrements of outflux (0.50 mmol) in the presence of ouabain and Na(o). However, these diuretics had minimal inhibitory effects on outflux in the absence of Na(o) suggesting that they inhibited principally the Na(o)-dependent outflux. Whereas this ouabain-uninhibited portion of outflux was dependent on Na(o), it was independent of K(o). Contrary to expectations, Na influx did not change when intracellular Na was altered. No uphill, net Na transport (ouabain-uninhibited) could be demonstrated under a variety of circumstances. Furosemide at high concentrations inhibited ATPase, reducing both ouabain-sensitive and ouabain-insensitive enzyme at 1.0 mM concentration while showing no effect on ATPase at 0.05-0.1 mM concentration. The effects of furosemide on ATPase and on Na flux were dissociable on a dose-response curve. Energy depletion for 22 h practically eliminated the Na(o)-dependent, diuretic-inhibited Na outflux. Activation energies and temperature coefficients for the diuretic-inhibited outflux were one-half the values for the classic ouabain-inhibited pump. These data are interpreted as evidence against a second Na pump. Exchange diffusion accounts adequately for most of these observations; however, the ouabain-insensitive fluxes may be complex and composed of several processes.