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Rehydration of high-density sickle erythrocytes in vitro.

Abstract

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.

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  • Authors+Show Affiliations

    ,

    Department of Aerospace Engineering, University of Cincinnati College of Engineering; the Cincinnati Comprehensive Sickle Cell Center, Cincinnati, OH 4526. -0508, USA.

    , , ,

    Source

    Blood 100:8 2002 Oct 15 pg 3017-25

    MeSH

    Anemia, Sickle Cell
    Bumetanide
    Cell Fractionation
    Erythrocytes
    Fluid Therapy
    Homozygote
    Humans
    In Vitro Techniques
    Phosphatidylserines

    Pub Type(s)

    Journal Article
    Research Support, U.S. Gov't, P.H.S.

    Language

    eng

    PubMed ID

    12351416

    Citation

    Holtzclaw, J David, et al. "Rehydration of High-density Sickle Erythrocytes in Vitro." Blood, vol. 100, no. 8, 2002, pp. 3017-25.
    Holtzclaw JD, Jiang M, Yasin Z, et al. Rehydration of high-density sickle erythrocytes in vitro. Blood. 2002;100(8):3017-25.
    Holtzclaw, J. D., Jiang, M., Yasin, Z., Joiner, C. H., & Franco, R. S. (2002). Rehydration of high-density sickle erythrocytes in vitro. Blood, 100(8), pp. 3017-25.
    Holtzclaw JD, et al. Rehydration of High-density Sickle Erythrocytes in Vitro. Blood. 2002 Oct 15;100(8):3017-25. PubMed PMID: 12351416.
    * Article titles in AMA citation format should be in sentence-case
    TY - JOUR T1 - Rehydration of high-density sickle erythrocytes in vitro. AU - Holtzclaw,J David, AU - Jiang,Maorong, AU - Yasin,Zahida, AU - Joiner,Clinton H, AU - Franco,Robert S, PY - 2002/9/28/pubmed PY - 2002/12/6/medline PY - 2002/9/28/entrez SP - 3017 EP - 25 JF - Blood JO - Blood VL - 100 IS - 8 N2 - 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. SN - 0006-4971 UR - https://www.unboundmedicine.com/medline/citation/12351416/Rehydration_of_high_density_sickle_erythrocytes_in_vitro_ L2 - http://www.bloodjournal.org/cgi/pmidlookup?view=long&amp;pmid=12351416 DB - PRIME DP - Unbound Medicine ER -