A PEROXIDATIVE MODEL OF HUMAN ERYTHROCYTE INTRACELLULAR CA2+ CHANGES WITH IN-VIVO CELL AGING - MEASUREMENT BY F-19-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 (F-19-NMR) to measure [Ca2+](i) following mild hydrogen peroxide (H2O2) stress to young red cells. Cells were separated using density centrifugation, exposed to 815 mu M 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.