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Blood, Vol. 92 No. 12 (December 15), 1998:
pp. 4844-4855
Two Distinct Pathways Mediate the Formation of Intermediate Density
Cells and Hyperdense Cells From Normal Density Sickle Red Blood
Cells
Robert S. Schwartz,
Sylvia Musto,
Mary E. Fabry, and
Ronald L. Nagel
From The Albert Einstein College of Medicine and Montefiore Medical
Center, Bronx Comprehensive Sickle Cell Center and Division of
Hematology, Bronx, NY.
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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