AJP - Cell Physiology, Vol 260, Issue 2 C308-C315, Copyright © 1991 by American Physiological Society
Swelling-activated K-Cl cotransport: metabolic dependence and inhibition by vanadate and fluoride
W. C. O'Neill
Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322.
Activation of K-Cl cotransport by cell swelling was studied by measuring K
influx in isotonic and hypotonic media in human red blood cells after
depletion of cellular ATP and after exposure to vanadate or fluoride.
Preincubation of red blood cells with 2-deoxyglucose resulted in an
inhibition of swelling-activated K-Cl cotransport that paralleled the
decline in cellular ATP. Subsequent repletion of ATP by incubation in
glucose, phosphate, and guanosine partially restored swelling-activated
K-Cl cotransport. Swelling-activated K-Cl cotransport was also inhibited by
200 microM vanadate. This inhibition was partially blocked by DIDS,
indicating an intracellular action, and required a 40-min preincubation,
suggesting that inhibition was due to vanadyl rather than vanadate.
Activation of K-Cl cotransport in swollen cells was also blocked by 16 mM
fluoride, an effect that was immediate and independent of Cl concentration.
Incubation of cells with 1 mM adenosine 3',5'-cyclic monophosphate (cAMP)
to raise intracellular cAMP levels did not inhibit swelling-activated K-Cl
cotransport, indicating that fluoride was not acting through adenyl
cyclase. No inhibition of Cl-dependent or bumetanide-sensitive K influx in
isotonic medium (Na-K-2Cl cotransport) was observed with ATP depletion,
vanadate, fluoride, or cAMP. Activation of K-Cl cotransport by
N-ethylmaleimide (NEM) was inhibited by ATP depletion but only partially
inhibited by fluoride and not inhibited by vanadate. Fluoride inhibited
K-Cl cotransport only when added before NEM treatment. These results
suggest that activation of K-Cl cotransport by cell swelling requires ATP
and involves a phosphohydrolase or phosphotransferase reaction that is
inhibited by vanadyl and fluoride.(ABSTRACT TRUNCATED AT 250 WORDS)
