Swelling-activated K-Cl cotransport: metabolic dependence and inhibition by vanadate and fluoride.
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)
O'Neill
W C
WC
Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322.
D000255:Adenosine Triphosphate / Q000378:metabolism; D002034:Bumetanide / Q000494:pharmacology; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000097:blood*; D002712:Chlorides / Q000097:blood*; D000242:Cyclic AMP / Q000097:blood / Q000494:pharmacology; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D005459:Fluorides / Q000494:pharmacology*; D006801:Humans; D006982:Hypertonic Solutions; D007038:Hypotonic Solutions; D066298:In Vitro Techniques; D007700:Kinetics; D009994:Osmolar Concentration; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000097:blood*; D027981:Symporters; D014638:Vanadates / Q000494:pharmacology*; D000096922:K Cl- Cotransporters
D000255:Adenosine Triphosphate; D002034:Bumetanide; D002352:Carrier Proteins; D002712:Chlorides; D000242:Cyclic AMP; D005033:Ethylmaleimide; D005459:Fluorides; D006982:Hypertonic Solutions; D007038:Hypotonic Solutions; D010042:Ouabain; D011188:Potassium; D027981:Symporters; D014638:Vanadates; D000096922:K Cl- Cotransporters