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Prepublished online as a Blood First Edition Paper on June 21, 2002; DOI 10.1182/blood-2002-02-0631.
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Blood, 15 October 2002, Vol. 100, No. 8, pp. 3017-3025
RED CELLS
Rehydration of high-density sickle erythrocytes in vitro
J. David Holtzclaw,
Maorong Jiang,
Zahida Yasin,
Clinton H. Joiner, and
Robert
S. Franco
From the Department of Aerospace Engineering,
University of Cincinnati College of Engineering; the Cincinnati
Comprehensive Sickle Cell Center; the Division of Hematology-Oncology,
University of Cincinnati College of Medicine; and the Division of
Hematology-Oncology, Children's Hospital Research Foundation;
Cincinnati, OH.
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
( > 1.107 g/cc) to either oxygenated incubation or rapid
oxygenated/deoxygenated (oxy/deoxy) cycling (70 seconds per
cycle). Light cells ( < 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.
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