Effects of oxidative stress on intact human erythrocytes were investigated using tert-butyl hydroperoxide (tBHP). Exposure of erythrocytes to tBHP caused a marked decrease in filterability in a time-dependent manner. Erythrocytes exposed to tBHP also show an increase in mean corpuscular volume and a remarkable formation of methemoglobin (met-Hb) without any appearance of hemichromes that form Heinz bodies. High performance liquid chromatography demonstrated that the tBHP-treated erythrocytes exhibited an apparent decrease in the membrane phospholipid, phosphatidylethanolamine (PE). The decrease in PE was inhibited by pretreatment with ascorbate, but not with verapamil. SDS-polyacrylamide gel electrophoresis of the tBHP-treated erythrocyte membrane showed a degradation of spectrin, band 3, band 4.2, and band 4.5, accompanied by the appearance of low-molecular-weight products. The degradation of the membrane proteins was not prevented by pretreatment with verapamil or ascorbate. However, the pretreatment with verapamil but not with ascorbate revealed significant inhibition of the tBHP-induced impairment in filterability in the presence of extracellular Ca2+. Thus, the present study shows that verapamil, a potent drug in reperfusion therapy, plays an important role in protection against oxidative injury, based on a close linkage among decreased filterability, met-Hb formation, and impaired membrane integrity.
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