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.
Garay
R P
RP
INSERM U7/CNRS UA 318, Hôpital Necker, Paris, France.
Nazaret
C
C
Hannaert
P A
PA
Cragoe
E J
EJ
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002034:Bumetanide / Q000494:pharmacology*; D002264:Carboxylic Acids / Q000494:pharmacology*; D002478:Cells, Cultured; D002712:Chlorides / Q000097:blood*; D004232:Diuretics / Q000494:pharmacology*; D004912:Erythrocytes / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D006801:Humans; D007192:Indenes / Q000494:pharmacology*; D011188:Potassium / Q000097:blood*; D012964:Sodium / Q000097:blood*
D002264:Carboxylic Acids; D002712:Chlorides; D004232:Diuretics; D007192:Indenes; D002034:Bumetanide; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D012964:Sodium; D005033:Ethylmaleimide; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D011188:Potassium