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Two distinct pathways mediate the formation of intermediate density cells and hyperdense cells from normal density sickle red blood cells.

Abstract

In sickle cell anemia (SS), some red blood cells dehydrate, forming a hyperdense (HD) cell fraction (>1.114 g/mL; mean corpuscular hemoglobin concentration [MCHC], >46 g/dL) that contains many irreversibly sickled cells (ISCs), whereas other SS red blood cells dehydrate to an intermediate density (ID; 1.090 to 1.114 g/mL; MCHC, 36 to 46 g/dL). This study asks if the potassium-chloride cotransporter (K:Cl) and the calcium-dependent potassium channel [K(Ca2+)] are participants in the formation of one or both types of dense SS red blood cells. We induced sickling by exposing normal density (ND; 1.080 to 1.090 g/mL; MCHC, 32 to 36 g/dL) SS discocytes to repetitive oxygenation-deoxygenation (O-D) cycles in vitro. At physiologic Na+, K+, and Cl-, and 0.5 to 2 mmol/L Ca2+, the appearance of dense cells was time- and pH-dependent. O-D cycling at pH 7.4 in 5% CO2-equilibrated buffer generated only ID cells, whereas O-D cycling at pH 6.8 in 5% CO2-equilibrated buffer generated both ID and HD cells, the latter taking more than 8 hours to form. At 22 hours, 35% +/- 17% of the parent ND cells were recovered in the ID fraction and 18% +/- 11% in the HD fraction. Continuous deoxygenation (N2/5% CO2) at pH 6.8 generated both ID and HD cells, but many of these cells had multiple projections, clearly different from the morphology of endogenous dense cells and ISCs. Continuous oxygenation (air/5% CO2) at pH 6.8 resulted in less than 10% dense cell (ID + HD) formation. ATP depletion substantially increased HD cell formation and moderately decreased ID cell formation. HD cells formed after 22 hours of O-D cycling at pH 6.8 contained fewer F cells than did ID cells, suggesting that HD cell formation is particularly dependent on HbS polymerization. EGTA chelation of buffer Ca2+ inhibited HD but not ID cell formation, and increasing buffer Ca2+ from 0.5 to 2 mmol/L promoted HD but not ID cell formation in some SS patients. Substitution of nitrate for Cl- inhibited ID cell formation, as did inhibitors of the K:Cl cotransporter, okadaic acid, and [(dihydroindenyl) oxy]alkanoic acid (DIOA). Conversely, inhibitors of K(Ca2+), charybdotoxin and clotrimazole, inhibited HD cell formation. The combined use of K(Ca2+) and K:Cl inhibitors nearly eliminated dense cell (ID + HD cell) formation. In summary, dense cells formed by O-D cycling for 22 hours at pH 7.4 cycling are predominately the ID type, whereas dense cells formed by O-D cycling for 22 hours at pH 6.8 are both the ID and HD type, with the latter low in HbF, suggesting that HD cell formation has a greater dependency on HbS polymerization. A combination of K:Cl cotransport and the K(Ca2+) activities account for the majority of dense cells formed, and these pathways can be driven independently. We propose a model in which reversible sickling-induced K+ loss by K:Cl primarily generates ID cells and K+ loss by the K(Ca2+) channel primarily generates HD cells. These results imply that both pathways must be inhibited to completely prevent dense SS cell formation and have potential therapeutic implications.

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  • Authors+Show Affiliations

    ,

    The Albert Einstein College of Medicine and Montefiore Medical Center, Bronx Comprehensive Sickle Cell Center and Division of Hematology, Bronx, NY, USA. rschwart@worldnet.att.net

    , ,

    Source

    Blood 92:12 1998 Dec 15 pg 4844-55

    MeSH

    Adenosine Triphosphate
    Anemia, Sickle Cell
    Buffers
    Calcium
    Carbon Dioxide
    Carrier Proteins
    Centrifugation, Density Gradient
    Chlorides
    Enzyme Inhibitors
    Erythrocytes
    Fetal Hemoglobin
    Humans
    Hydrogen-Ion Concentration
    Ion Transport
    Oxygen
    Potassium
    Specific Gravity
    Symporters
    Time Factors

    Pub Type(s)

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

    Language

    eng

    PubMed ID

    9845552

    Citation

    Schwartz, R S., et al. "Two Distinct Pathways Mediate the Formation of Intermediate Density Cells and Hyperdense Cells From Normal Density Sickle Red Blood Cells." Blood, vol. 92, no. 12, 1998, pp. 4844-55.
    Schwartz RS, Musto S, Fabry ME, et al. Two distinct pathways mediate the formation of intermediate density cells and hyperdense cells from normal density sickle red blood cells. Blood. 1998;92(12):4844-55.
    Schwartz, R. S., Musto, S., Fabry, M. E., & Nagel, R. L. (1998). Two distinct pathways mediate the formation of intermediate density cells and hyperdense cells from normal density sickle red blood cells. Blood, 92(12), pp. 4844-55.
    Schwartz RS, et al. Two Distinct Pathways Mediate the Formation of Intermediate Density Cells and Hyperdense Cells From Normal Density Sickle Red Blood Cells. Blood. 1998 Dec 15;92(12):4844-55. PubMed PMID: 9845552.
    * Article titles in AMA citation format should be in sentence-case
    TY - JOUR T1 - Two distinct pathways mediate the formation of intermediate density cells and hyperdense cells from normal density sickle red blood cells. AU - Schwartz,R S, AU - Musto,S, AU - Fabry,M E, AU - Nagel,R L, PY - 1998/12/9/pubmed PY - 1998/12/9/medline PY - 1998/12/9/entrez SP - 4844 EP - 55 JF - Blood JO - Blood VL - 92 IS - 12 N2 - In sickle cell anemia (SS), some red blood cells dehydrate, forming a hyperdense (HD) cell fraction (>1.114 g/mL; mean corpuscular hemoglobin concentration [MCHC], >46 g/dL) that contains many irreversibly sickled cells (ISCs), whereas other SS red blood cells dehydrate to an intermediate density (ID; 1.090 to 1.114 g/mL; MCHC, 36 to 46 g/dL). This study asks if the potassium-chloride cotransporter (K:Cl) and the calcium-dependent potassium channel [K(Ca2+)] are participants in the formation of one or both types of dense SS red blood cells. We induced sickling by exposing normal density (ND; 1.080 to 1.090 g/mL; MCHC, 32 to 36 g/dL) SS discocytes to repetitive oxygenation-deoxygenation (O-D) cycles in vitro. At physiologic Na+, K+, and Cl-, and 0.5 to 2 mmol/L Ca2+, the appearance of dense cells was time- and pH-dependent. O-D cycling at pH 7.4 in 5% CO2-equilibrated buffer generated only ID cells, whereas O-D cycling at pH 6.8 in 5% CO2-equilibrated buffer generated both ID and HD cells, the latter taking more than 8 hours to form. At 22 hours, 35% +/- 17% of the parent ND cells were recovered in the ID fraction and 18% +/- 11% in the HD fraction. Continuous deoxygenation (N2/5% CO2) at pH 6.8 generated both ID and HD cells, but many of these cells had multiple projections, clearly different from the morphology of endogenous dense cells and ISCs. Continuous oxygenation (air/5% CO2) at pH 6.8 resulted in less than 10% dense cell (ID + HD) formation. ATP depletion substantially increased HD cell formation and moderately decreased ID cell formation. HD cells formed after 22 hours of O-D cycling at pH 6.8 contained fewer F cells than did ID cells, suggesting that HD cell formation is particularly dependent on HbS polymerization. EGTA chelation of buffer Ca2+ inhibited HD but not ID cell formation, and increasing buffer Ca2+ from 0.5 to 2 mmol/L promoted HD but not ID cell formation in some SS patients. Substitution of nitrate for Cl- inhibited ID cell formation, as did inhibitors of the K:Cl cotransporter, okadaic acid, and [(dihydroindenyl) oxy]alkanoic acid (DIOA). Conversely, inhibitors of K(Ca2+), charybdotoxin and clotrimazole, inhibited HD cell formation. The combined use of K(Ca2+) and K:Cl inhibitors nearly eliminated dense cell (ID + HD cell) formation. In summary, dense cells formed by O-D cycling for 22 hours at pH 7.4 cycling are predominately the ID type, whereas dense cells formed by O-D cycling for 22 hours at pH 6.8 are both the ID and HD type, with the latter low in HbF, suggesting that HD cell formation has a greater dependency on HbS polymerization. A combination of K:Cl cotransport and the K(Ca2+) activities account for the majority of dense cells formed, and these pathways can be driven independently. We propose a model in which reversible sickling-induced K+ loss by K:Cl primarily generates ID cells and K+ loss by the K(Ca2+) channel primarily generates HD cells. These results imply that both pathways must be inhibited to completely prevent dense SS cell formation and have potential therapeutic implications. SN - 0006-4971 UR - https://www.unboundmedicine.com/medline/citation/9845552/Two_distinct_pathways_mediate_the_formation_of_intermediate_density_cells_and_hyperdense_cells_from_normal_density_sickle_red_blood_cells_ L2 - http://www.bloodjournal.org/cgi/pmidlookup?view=long&pmid=9845552 DB - PRIME DP - Unbound Medicine ER -