Effect of metabolic depletion on the furosemide-sensitive Na and K fluxes in human red cells

Summary We report in this paper the effect of metabolic depletion on several modes of furosemide-sensitive (FS) Na and K transport in human red blood cells. The reduction of ATP content below 100 mol/liter cells produced a marked decrease in the maximal activation (V_max) of the outward. FS transport of Na and K into choline medium in the presence of ouabain (0.1 mM) and 1 mM MgCl₂. TheK_0.5 for internal Na to activate the FS Na efflux was not altered by metabolic depletion. However, metabolic depletion markedly decreased the K_i for external K (K_o) to inhibit the FS Na efflux into choline medium (from 25 to 11 mM). Repletion of ATP content by incubation of cells in a substraterich medium recovered control levels ofV_max of the FS Na and K fluxes and of K_i for external K to inhibit FS Na efflux. TheV_max of FS Na and K influxes was also markedly decreased when the ATP content dropped below 100 mol/liter cells. This was mainly due to a decrease in the inward-coupled transport of K and Na (Na_o-stimulated K influx and the K_o-stimulated Na influx). The FS K_i/K_o exchange pathway of the Na–K cotransport, estimated from the FS K influx from choline-20 mM K_o medium into cells containing 22 mmol Na/liter cells, was also reduced by starvation. Starvation did not inhibit the FS Na_i/Na_o exchange pathway, estimated as FS Na influx from a medium containing 130 mM NaCl into cells containing 22 mmol Na/liter cells. The unidirectional FS²²Na efflux and influx were also measured in control and starved cells containing 22 mmol Na/liter cells, incubated in a Na medium (130 mM) at varying external K (0 to 20 mM). In substrate-fed cells, incubated in the absence of external K, FS Na efflux was larger than Na influx. This FS net Na extrusion (400 to 500 mol/liter cells·hr) decreased when external K was increased, approaching zero around 15 mM K_o. In starved cells the net Na extrusion was markedly decreased and it approached zero at lower K_o than in substrate-fed cells. Our results indicate that the FS Na and K fluxes, and their major component, the gradient driven Na–K–Cl cotransport system, are dependent on the metabolic integrity of the cells.