Abstract
KCl Cotransport (KCC) is active in normal (AA) reticulocytes and overly active in sickle (SS) reticulocytes. Cell swelling activates KCC and induces a powerful regulatory volume decrease (RVD) in reticulocytes, which increases cellular hemoglobin concentration (CHC) to new steady state values that are higher in SS than AA cells (Blood2004;104(9):2954–60). We recently showed that urea (300–900 mM), which strongly activates KCC, also induces an intense RVD with even higher final CHC values (SS>AA) (Blood2004; 104 (11): 976a). Because KCC activity is high in reticulocyte-rich samples in both SS and AA blood, KCC activity has been assumed to be minimal in mature cells. We now report that mature RBC exhibit RVD stimulated by urea and mediated by KCC. AA and SS RBC were washed in HBS and treated with nystatin to increase cation content and decrease CHC to 22–24 gm/dl. During incubation at 37o in HBS (145 mM NaCl, 5 KCl, 1 MgCl2, 10 glucose, 20 HEPES, pH 7.4) ± 600 mM urea, timed samples were taken into iced HBS, washed, and kept on ice until analyzed later that day on an Advia 120 automated cell counter, which reports frequency distributions for CHC of both mature RBC and reticulocytes. As previously reported, within 30 min reticulocytes achieved a new steady state CHC which was higher for SS than AA cells, though the speed of RVD was similar. Surprisingly, mean CHC of mature (non-reticulocyte) RBC in both AA and SS blood also increased upon incubation with urea. RVD in mature cells was slower than in reticulocytes and was apparently incomplete after 2 hours. RVD in mature RBC was completely abrogated (CHC was stable) in the absence of Cl- (sulfamate substitution) or in the presence of 100 uM DIOA (dihydro-indenyl-oxy-alkanoic acid), both of which inhibit KCC activity. Whereas reticulocyte CHC frequency distributions after urea-stimulated KCC-mediated RVD showed a single population, CHC distributions for mature RBC revealed two distinct sub-populations: One in which CHC changed little during incubation and a second which achieved a CHC similar to that achieved by reticulocytes after RVD. The relative size of the volume regulating (high CHC) sub-population increased steadily throughout the incubation, which was responsible for the progressive increase in mean CHC values. The high CHC sub-population was not apparent when cells were incubated in Cl- free media or with DIOA, indicating that RVD was mediated by KCC. After 2 hours incubation, 67 ± 8 % of SS RBC had shifted to higher CHC, compared to 37 ± 11 % of mature AA RBC (p<<0.001 by t-test). The progressive change in CHC histograms during incubation was consistent with cells achieving the same final CHC values at various rates. In preliminary studies with biotin-labeled AA cells ageing in vivo, urea-stimulated RVD in mature cells diminished with time, but persisted through most of RBC lifespan. These data indicate that the KCl cotransporter remains in the membrane of mature AA RBC, and is capable of producing RVD under the strong stimulation of urea. In SS RBC, which have shorter lifespan, a majority of non-reticulocytes retain urea-stimulated KCC activity.
