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

Abstract

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 mumol/liter cells produced a marked decrease in the maximal activation (Vmax) of the outward, FS transport of Na and K into choline medium in the presence of ouabain (0.1 mM) and 1 mM MgCl2. The K0.5 for internal Na to activate the FS Na efflux was not altered by metabolic depletion. However, metabolic depletion markedly decreased the Ki for external K (Ko) to inhibit the FS Na efflux into choline medium (from 25 to 11 mM). Repletion of ATP content by incubation of cells in a substrate-rich medium recovered control levels of Vmax of the FS Na and K fluxes and of Ki for external K to inhibit FS Na efflux. The Vmax of FS Na and K influxes was also markedly decreased when the ATP content dropped below 100 mumol/liter cells. This was mainly due to a decrease in the inward-coupled transport of K and Na (NaO-stimulated K influx and the Ko-stimulated Na influx). The FS Ki/Ko exchange pathway of the Na-K cotransport, estimated from the FS K influx from choline-20 mM Ko medium into cells containing 22 mmol Na/liter cells, was also reduced by starvation. Starvation did not inhibit the FS Nai/Nao exchange pathway, estimated as FS Na influx from a medium containing 130 mM NaCl into cells containing 22 mmol Na/liter cells. The unidirectional FS 22Na 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 mumol/liter cells X hr) decreased when external K was increased, approaching zero around 15 mM Ko. In starved cells the net Na extrusion was markedly decreased and it approached zero at lower Ko 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.