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Generation of light sickle erythrocytes from dense sickle erythrocytes during fast oxy/deoxy cycling

, : Generation of light sickle erythrocytes from dense sickle erythrocytes during fast oxy/deoxy cycling. Blood 96(11 Part 1): 601a, November 16

Recent studies have identified older, low-density sickle red blood cells (SS RBC) that are resistant to dehydration mediated by valinomycin or the Ca-activated K channel (K(Ca)). We hypothesize that these cells derive from dense SS RBC that become rehydrated. To study rehydration we used a dual channel, fast cycle apparatus to simulate in vivo oxy/deoxy conditions. A 1% suspension of dense (>1.107 g/cc) SS RBC was cycled through gas permeable silicone tubing in a continuous, closed circuit. Each channel contained deoxygenator and oxygenator sections in which pO2 was measured continuously (35.0+-8.6 mmHg and 71.8+-15.6 mmHg). Circuit time was 72.8+-2.0 seconds and total cycling time was 97.4+-10.9 minutes. After cycling, the cells were separated by density and the generation of light cells (<1.087 g/cc) quantified. Statistical comparison were paired t-tests with concurrent controls. There was some light cell generation during static incubations at 37C (1.6+-1.2%, n=8). Fast oxy/deoxy cycling produced a significant increase in light cell formation (9.5+-5.7%, n=8, p<0.004). The rehydrated cells were K depleted (22.7+-21.3 mM/KgHb, n=5) and Na loaded (368.1+-100.8 mM/KgHb, n=5). The presence of 1.5 mM external Ca++ significantly inhibited the formation of rehydrated SS RBC (4.3+-2.8% with Ca++ vs. 9.5+-5.7% without, n=8, p<0.02) during oxy/deoxy cycling. Pre-incubation of the dense cells with 45 mM DIDS, an inhibitor of deoxygenation-induced (DI) cation flux and chloride conductance, significantly inhibited the generation of light cells during fast oxy/deoxy cycling (2.2+-1.0% with DIDS vs. 3.6+-1.3% without, n=7, p<0.004). Finally, the presence during cycling of 1 mM bumetanide, which inhibits increased cation fluxes in light sickle cells, also decreased light cell formation (4.6+-2.6% with bumetanide vs. 7.2+-3.9% without, n=7, p<0.029). These findings support the hypothesis that older, low-density SS RBC derive from dense SS RBC. Inhibition of light cell generation by DIDS suggests that the DI pathway, previously implicated in SS RBC dehydration, may also be involved in rehydration of dense, K depleted cells. Inhibition of light cell generation by Ca++ is consistent with the reduction of DI Na influx by Ca++ reported previously. Alternatively, Ca++ inhibition of cycling-dependent SS RBC rehydration may represent a competition between rehydrating pathway(s) and the K(Ca) dehydrating pathway.


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