Stimulation of erythrocyte phosphatidylserine exposure by lead ions

Abstract

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.