Rehydration of high-density sickle erythrocytes in vitro.Blood 2002; 100(8):3017-25Blood
Recent studies have identified older, low-density sickle red blood cells (SSRBCs) that were resistant to dehydration by valinomycin, a K(+) ionophore. These cells, thought to derive from dense SSRBCs that have rehydrated, may represent a terminal cellular phase. To study rehydration, we subjected dense SSRBCs (rho > 1.107 g/cc) to either oxygenated incubation or rapid oxygenated/deoxygenated (oxy/deoxy) cycling (70 seconds per cycle). Light cells (rho < 1.087 g/cc) were generated during both oxy incubation (2.9% +/- 2.1%; n = 42) and oxy/deoxy cycling (5.3% +/- 2.4%; n = 42). The rehydrated cells were K(+)-depleted (K(+) = 20 +/- 14 mmol/kg hemoglobin [Hb]) and Na(+)-loaded (Na(+) = 394 +/- 106 mmol/kg Hb), and had high levels of external phosphatidylserine. In the presence of external calcium, the generation of rehydrated SSRBCs was inhibited during oxy/deoxy cycling, but the percentage with external phosphatidylserine increased. The calcium-mediated inhibition of rehydration was reversed by charybdotoxin, implying that rehydration was delayed in some cells by the Ca(++)-activated K(+) channel. Preincubation of dense SSRBCs with DIDS (4,4'-di-isothiocyanato-2,2'-disulfostilbene) inhibited the generation of light cells during fast oxy/deoxy cycling, but not during oxy incubation. These results suggest that the sickling-induced pathway, previously implicated in SSRBC dehydration, may be involved in the deoxy-dependent component of rehydration for dense, K(+)-depleted cells. Light-cell generation was inhibited by 1 mM bumetanide during both oxy incubation and oxy/deoxy cycling, providing evidence that a bumetanide-sensitive, deoxy-independent pathway, previously described in circulating light SSRBCs, also contributes to the rehydration of high-density SSRBCs.