A peroxidative model of human erythrocyte intracellular Ca2+ changes with in vivo cell aging: measurement by 19F-NMR spectroscopy.

Numerous changes occur with human erythrocyte aging in vivo, including an increase in free ionic intracellular calcium concentration ([Ca2+]i) (N.R. Aiken et al. (1992) Biochim. Biophys. Acta 1136, 155-160). An attractive hypothesis of cell aging suggests that oxidative stress is responsible for many age-related changes. To determine whether oxidative stress leads to increased intracellular Ca2+ concentrations, we used the fluorinated calcium probe 5,5'-difluoroBAPTA and fluorine nuclear magnetic resonance spectroscopy (19F-NMR) to measure [Ca2+]i following mild hydrogen peroxide (H2O2) stress to young red cells. Cells were separated using density centrifugation, exposed to 815 microM H2O2, loaded with the calcium probe, and [Ca2+]i measured. Intracellular [Ca2+] increased from 62 nM (+/- 4, S.E.) in untreated young cells to 173 nM (+/- 11) in peroxide treated cohort young cells. This value approached our previously reported [Ca2+]i of 221 nM (+/- 25) in old human erythrocytes. Pretreatment of young cells with (a) cobalt, which blocks Ca2+ influx through calcium channels, or (b) carbon monoxide, which prevents methemoglobin formation, inhibited the peroxide-induced increase in ionic intracellular calcium. These findings are consistent with the hypothesis that oxidative stress of erythrocytes contributes to the increased [Ca2+]i found in senescent cells, and that this is due to increased membrane Ca2+ leak resulting from oxidatively induced methemoglobin-cytoskeletal protein crosslinking.