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Prepublished online as a Blood First Edition Paper on June 21, 2002; DOI 10.1182/blood-2002-02-0631.
Submitted February 27, 2002
Accepted May 29, 2002
Rehydration of high-density sickle erythrocytes in vitro
J D Holtzclaw, Maorong Jiang, Zahida Yasin, Clinton H Joiner, and Robert S Franco*
Department of Aerospace Engineering, University of Cincinnati College of Engineering, Cincinnati, OH, USA; Cincinnati Comprehensive Sickle Cell Center, Cincinnati, OH, USA; Division of Hematology-Oncology, University of Cincinnati College of Medicine, Cincinnati, OH, USA
Cincinnati Comprehensive Sickle Cell Center, Cincinnati, OH, USA; Division of Hematology-Oncology, Children's Hospital Research Foundation, Cincinnati, OH, USA
Cincinnati Comprehensive Sickle Cell Center, Cincinnati, OH, USA; Division of Hematology-Oncology, University of Cincinnati College of Medicine, Cincinnati, OH, USA
* Corresponding author; email: robert.franco{at}uc.edu.
Recent studies have identified older, low density sickle red blood cells (SSRBC) that were resistant to dehydration by valinomycin, a K+ ionophore. These cells, thought to derive from dense SSRBC that have rehydrated, may represent a terminal cellular phase. To study rehydration, we subjected dense SSRBC ( >1.107 g/cc) to either oxygenated incubation or rapid oxy/deoxy cycling (70 sec/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/KgHb) and Na+-loaded (Na+ = 394 ± 106 mmol/KgHb), and had high levels of external phosphatidylserine. In the presence of external calcium, the generation of rehydrated SSRBC 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. Pre-incubation of dense SSRBC with DIDS 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 SSRBC, also contributes to the rehydration of high density SSRBC.
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