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GROWTH, DIFFERENTIATION, AND APOPTOSIS

Stimulation of erythrocyte phosphatidylserine exposure by lead ions

Department of Physiology, University of Tübingen, Tübingen, Germany

Submitted 1 March 2004 ; accepted in final form 15 September 2004

Pb⁺ intoxication causes anemia that is partially due to a decreased life span of circulating erythrocytes. As shown recently, a Ca²⁺-sensitive erythrocyte scramblase is activated by osmotic shock, oxidative stress, and/or energy depletion, leading to exposure of phosphatidylserine at the erythrocyte surface. Because macrophages are equipped with phosphatidylserine receptors, they bind, engulf, and degrade phosphatidylserine-exposing cells. The present experiments were performed to explore whether Pb⁺ ions trigger phosphatidylserine exposure of erythrocytes. The phosphatidylserine exposure was estimated on the basis of annexin binding as determined using fluorescence-activated cell sorting (FACS) analysis. Exposure to Pb⁺ ions [0.1 µM Pb(NO₃)₂] significantly increased annexin binding. This effect was paralleled by erythrocyte shrinkage, which was apparent on the basis of the decrease in forward scatter in FACS analysis. The effect of Pb⁺ ions on cell volume was virtually abolished, and the effect of Pb⁺ ions on annexin binding was blunted after increase of extracellular K⁺ concentration. Moreover, both effects of Pb⁺ ions were partially prevented in the presence of clotrimazole (10 µM), an inhibitor of the Ca²⁺-sensitive K⁺ channels in the erythrocyte cell membrane. Whole cell patch-clamp experiments disclosed a significant activation of a K⁺-selective conductance after Pb⁺ ion exposure, an effect requiring higher (10 µM) concentrations, however. In conclusion, Pb⁺ ions activate erythrocyte K⁺ channels, leading to erythrocyte shrinkage, and also activate the erythrocyte scramblase, leading to phosphatidylserine exposure. The effect could well contribute to the reported decreased life span of circulating erythrocytes during Pb⁺ intoxication.

cell volume; annexin; apoptosis; Gardos channel; calcium

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