Decreased magnesium effects of echinocytes formed by calcium plus A23187 in cryopreservation

Echinocytes appeared in thawed-washed erythrocytes previously frozen with glycerol in response to Ca2+ plus divalent cation ionophore A23187. The occurrence of echinocytes depended on the concentrations of Ca2+. When the incidence of echinocytes was plotted against the log dose of Ca2+, a sigmoidal curve was obtained and fitted the probit plots well. ED50 values for frozen-thawed and fresh erythrocytes were 38 +/- 10 and 40 +/- 9 microM (mean +/- SD) CaCl2, respectively. The effects of divalent cations, such as Co2+, Mn2+, Ba2+, and Mg2+, on echinocyte formation were examined. Increasing Mg2+ concentrations only shifted the dose-response curve parallel to the right side. However, in cryopreserved erythrocytes in the medium with high Mg2+ content, the degree of the shift was less than that of fresh erythrocytes. ED50 values in 2 mM Mg2+ medium for frozen-thawed and fresh erythrocytes were 62 +/- 15 and 130 +/- 12 microM CaCl2, respectively. Under this condition, the lowest adenosine triphosphate (ATP) levels of about 60% of the control level were established at Ca2+ concentrations in cryopreserved erythrocytes lower than those in fresh ones. Then the utilization of cellular ATP decreased with echinocyte formation. These results indicate that futile hydrolysis of ATP in cryopreserved erythrocytes in high Mg2+ media may reduce Ca2+ excretion by activation of the Ca2+ pump in plasma membranes, resulting in echinocyte formation in low Ca2+ concentration.