Cation transport in oxidant-stressed human erythrocytes: heightened N-ethylmaleimide activation of passive K+ influx after mild peroxidation.
Normal and chronically dehydrated (hereditary xerocytosis) human red cells were subjected to mild peroxidative treatment (315 microM hydrogen peroxide (H2O2), 15 min) in the presence of azide. The subsequent expression of passive (ouabain-resistant) K+ transport activities was analyzed by measurement of 86Rb+ influx. Peroxidation of normal red cells did not affect basal K+ transport activity, but the increment in K+ influx elicited by 0.5 mM N-ethylmaleimide (NEM) was increased 3-fold. The enhanced K+ influx was chloride-dependent, but only partially inhibited by 0.1 mM furosemide. Stimulated activity declined progressively after NEM activation, but could be restored by a second NEM treatment. Prior conversion of hemoglobin to the carbonmonoxy form abolished the response to peroxide, while 200 microM butylated hydroxytoluene (BHT) exerted only partial inhibition, suggesting that the effect of H2O2 requires interaction of activated, unstable hemoglobin species with the membrane, but that lipid peroxidation is not sufficient. Peroxidation following NEM treatment also enhanced NEM activation, indicating that enhancement does not require altered NEM reactions with stimulatory or inhibitory sites. Passive K+ transport in hereditary xerocytosis red cells was not activated by NEM, with or without H2O2 pretreatment. The results demonstrate that modest peroxidative damage to red cells can heighten the activation of a transport system that is thought to be capable of mediating net K+ efflux and volume reduction in cells that express it. Models are proposed in which the effects of NEM, H2O2, cell swelling and other factors are mediated by conformational changes in a postulated subpopulation of anion channel (Band 3) molecules that bind the K+ transporter.
Sheerin
H E
HE
Cell Biology Group, Worcester Foundation for Experimental Biology, Shrewsbury, MA.
Snyder
L M
LM
Fairbanks
G
G
D002084:Butylated Hydroxytoluene / Q000494:pharmacology; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology*; D006402:Hematologic Diseases / Q000097:blood / Q000235:genetics; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007700:Kinetics; D011188:Potassium / Q000097:blood*; D012016:Reference Values; D012413:Rubidium / Q000097:blood
D002084:Butylated Hydroxytoluene; D006861:Hydrogen Peroxide; D012413:Rubidium; D005033:Ethylmaleimide; D011188:Potassium