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

In sickle cell anemia (SS), some red blood cells dehydrate, forming a hyper dense (HD) cell fraction (>1.114 g/mL; mean corpuscular hemoglobin concentr ation [MCHC], >46 g/dL) that contains many irreversibly sickled cells (ISCs ), whereas other SS red blood cells dehydrate to an intermediate density (I D; 1.090 to 1.114 g/mL; MCHC, 36 to 46 g/dL). This study asks if the potass ium-chloride cotransporter (K:Cl) and the calcium-dependent potassium chann el [K(Ca2+)] are participants in the formation of one or both types of dens e 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 oxygen ation-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-d ependent. O-D cycling at pH 7.4 in 5% CO2-equilibrated buffer generated onl y ID cells, whereas O-D cycling at pH 6.8 in 5% CO2-equilibrated buffer gen erated both ID and HD cells, the latter taking more than 8 hours to form. A t 22 hours, 35% +/- 17% of the parent ND cells were recovered in the ID fra ction and 18% +/- 11% in the HD fraction. Continuous deoxygenation (N-2/5% 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 d ense cells and ISCs. Continuous oxygenation (air/5% CO2) at pH 6.8 resulted in less than 10% dense cell (ID + HD) formation. ATP depletion substantial ly 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 c ells than did ID cells, suggesting that HD cell formation is particularly d ependent 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, inhibi ted HD cell formation. The combined use of K(Ca2+) and K:Cl inhibitors near ly 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 a re 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 combinatio n of K:Cl cotransport and the K(Ca2+) activities account for the majority o f dense cells formed, and these pathways can be driven independently. We pr opose a model in which reversible sickling-induced K+ loss by K:Cl primaril y 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 comp letely prevent dense SS cell formation and have potential therapeutic impli cations. (C) 1998 by The American Society of Hematology.