Different sensitivities of rat and human red cells to exogenous Ca2+

Abstract

During an examination of the effects of shear and of the Ca2+ ionophore A23187 on Ca2+ entry into erythrocytes of rats and humans, we noted that rat erythrocytes were much more sensitive to Ca2+-induced hemolysis than the human cells. An examination of the effect of Ca2+ on transglutaminase, a cytosolic enzyme in the erythrocyte which cross-links membrane proteins and renders cells less deformable, demonstrated a correlation between enzyme activity and Ca2+-induced hemolysis. Both rat and human cells subjected to shear-induced Ca2+ entry exhibited increased enzyme activity and altered membrane protein SDS-PAGE patterns. Twenty micromolar A23187 with Ca2+ at concentrations above 80 microM caused hemolysis of rat erythrocytes. In contrast to human erythrocytes, under these conditions no membranes were recoverable from rat erythrocytes. At lower concentrations of Ca2+ (25 and 50 microM), however, rat erythrocytes maintained integrity, and exhibited enhanced transglutaminase activity and cross-linking of membrane proteins. The rat enzyme can be activated 30% by 10 microM Ca2+, while 50 microM Ca2+ was necessary to achieve a similar activation of the enzyme from human red blood cells. In studies of shear-stimulated Ca2+ uptake by erythrocytes the rat red cell enzyme was more readily activated. The SDS-PAGE pattern of rat red cell membranes after a 30 sec shear showed specific changes in protein banding, including the appearance of bands greater than 330 kDa. Changes in protein banding were also apparent in cytosolic proteins. This work supports the view that shear-induced Ca2+ entry activates transglutaminase that leads to cross-linking of membrane components, a loss of cell integrity, and eventual cell death.