Furosemide‐sensitive potassium efflux in cultured mouse fibroblasts

Abstract

Transfer of LM(TK-) cells from normal growth medium to medium lacking K+ leads to a rapid loss of intracellular K+, which is 50-70% inhibited by furosemide or bumetanide. The diuretic-sensitive component of K+ efflux requires both Na+ and Cl-, and is presumably mediated by a K+, Na+, Cl- cotransport system of the kind described in avian erythrocytes and Ehrlich ascites cells. It can be calculated that such a system should be near equilibrium under normal growth conditions but should mediate net efflux (as observed) when the driving force is altered by reducing extracellular K+. The diuretic-sensitive component of net K+ efflux is also sensitive to amiloride. This effect is probably indirect, however, with amiloride acting to block the Na+ influx that supplies Na+ to the cotransport system. At the low extracellular K+ concentrations employed in these studies, the diuretic-sensitive system is a physiologically important pathway of K+ loss. The rate of growth in low-K+ medium can be increased (or the rate of cell lysis decreased) by adding diuretic or by reducing external Na+ or Cl-.

Kinetic mechanism of Na+, K+, Cl−-cotransport as studied by Rb+ influx into HeLa cells: Effects of extracellular monovalent ions

“…We also obtained results indicating correlations of transmembrane gradients of Na § K+(Rb § and C1 with furosemide-sensitive Rb § influx (data not shown). The sum of the transmembrane gradients of the chemical potentials of Na § K § and CI-is generally accepted to be important in the cotransport system (Schmidt & McManus, 1977;Duhm & G6bel, 1984a;Jayme et al, 1984). Therefore, the implication of the effects of intracellular ions seems reasonable and it is premature to complete this transport model until the roles of in- (A.14)…”

“…Other anions have been reported to be unable to replace Cl-in supporting diuretic-sensitive or C1 -dependent cation fluxes (Chipperfield, 1980;Aiton et al, 1981;Aiton & Simmons, 1983;Jayme et al, 1984), except Br-, which is very slightly effective (Geck et al, 1980). Replacement of C1-in the medium by other anions changes the apparent Km for CI-in relation to diuretic-sensitive 86Rb+ influx into human red blood cells (Chipperfield, 1984) and HeLa cells (Tivey et al, 1985); nitrate and thiocyanate or isethionate in particular shift the Km toward higher concentrations than gluconate.…”

“…There are many reports on the kinetic characteristics and physiological roles of the diuretic-sensitive systems of mammalian cells including cultured cells (Gargus & Slayman, 1980;Koenig et al, 1983;Duhm & G6bel, 1984a,b;Jayme et al, 1984;Owen, 1984;Tivey et al, 1985). As the total net charge of the ions moved by the diuretic-sensitive systems is usually zero and the transport activity of the systems is not influenced by changes in membrane potential (Geck et al, 1980;McRoberts et al, 1982), the systems appear to be electrically silent.…”

“…An appreciable part of Na + or K + transport depends on extracellular C1-and is also linked to Cl-transport. Substitution of another monovalent anion for C1-has been reported to reduce Na + or K + flux in mammalian cells (Geck et al, 1980;Aiton et al, 1981;Chipperfield, 1981;Jayme et al, 1984). However, the loop-diuretics do not have synergistic effects with C1 deficiency in reducing cation transport (Aiton et al, 1981;Chipperfield, 1981).…”

Sodium 22+ washout from cultured rat cells

“…The bumetanide-sensitive Naf and K+ washout is considered to represent the extrusion component of Na+ , K+-cotranspoq (Jayme et al, 1984). To examine whether this transport system exists in cultured rat fibroblasts, we performed the washout experiments with or without .1 mM bumetanide in the medium (Fig.…”