Demonstration of a [K+,Cl-]-cotransport System in Human Red Cells by Its Sensitivity to [(dihydroindenyl)oxy]alkanoic Acids: Regulation of Cell Swelling and Distinction from the Bumetanide-sensitive [Na+,K+,Cl-]-cotransport System

A screening of several families of compounds on NEM-stimulated K+ efflux in human red cells allowed us to select a [(dihydroindenyl)oxy] alkanoic acid (DIOA) as the first potent inhibitor of this K+ flux (IC50 of 10(-5) M) without side effects on the bumetanide-sensitive [Na+,K+,Cl-]-cotransport system. Incubation of human red cells in hypotonic media (179 mosm) increased cell volume (by 18-20%) and provoked the appearance of a DIOA-sensitive K+ efflux of 4.48 +/- 0.83 mmol.(liter of cells X hr)-1 (mean +/- SD of nine experiments). This DIOA-sensitive K+ efflux exhibited a Michaelian-like dependence on the Cl- concentration of the incubation media (freely equilibrated with intracellular Cl-) with an apparent dissociation constant of 39.6 +/- 14.7 mM and a maximal rate of 4.7 +/- 0.9 mmol.(liter of cells X hr)-1 (mean +/- SD of five experiments). The chloride effect was mediated by intracellular and not by extracellular Cl-, as expected for an outward [K+,Cl-]-cotransport. The above properties of DIOA-sensitive K+ efflux clearly confirm that human red cells have a [K+,Cl-]-cotransport system that regulates cell swelling. The regulatory response to hypotonic media was also strongly depressed by cytochalasin B at a concentration of 1 mM, suggesting that the activating signal is probably transduced by the cytoskeleton.