Urea stimulation of KCl cotransport induces abnormal volume reduction in sickle reticulocytes

Abstract

KCl cotransport (KCC) activity contributes to pathologic dehydration in sickle (SS) red blood cells (RBCs). KCC activation by urea was measured in SS and IntroductionThe KCl cotransporter (KCC) mediates electroneutral coupled transport of K ϩ and Cl Ϫ in a variety of cell types. 1,2 KCC activation results in net efflux of KCl from cells with high potassium content, with accompanying water loss and volume reduction. KCC is active in reticulocytes and diminishes with red cell maturation. 3,4 It is thought to function physiologically to reduce red blood cell (RBC) volume and establish the high cellular hemoglobin concentration (CHC) characteristic of mature cells. KCC is activated in vitro by cell swelling, acid pH, sulfhydryl oxidation/alkylation, and exposure to urea, but the relative importance of these stimuli in vivo is unknown.In RBCs containing sickle hemoglobin (Hb S), KCC activity is high, in part because of the high percentage of reticulocytes. 4 However, Hb S or Hb C in trait RBCs (AS or AC) with normal mean cell age, or after incorporation into normal RBC ghosts, appears to increase KCC activity. 5,6 Expression of Hb S or C in transgenic mice also increases red cell KCC activity. 7 Recently, we demonstrated an abnormal response to acid pH in SS RBCs that was partially corrected by incubation with the sulfhydryl reducing agent, dithiothreitol. 8 These findings suggested that regulation of KCC is abnormal in SS RBCs.As a volume regulator, KCC activity is responsive to CHC. 9,10 KCC is activated on cell swelling, and, as KCl is lost, cell volume falls, cellular hemoglobin concentration increases, and transport activity diminishes. The resultant CHC thus reflects the physiologic "volume set point" of the system. The inactivation of KCC as the cell shrinks toward its new steady state is most likely a key factor in defining the volume regulatory function of the transporter, which in turn is crucial for establishing the steady state CHC for RBCs in vivo.Volume reduction mediated by KCC has been demonstrated with RBC density measurements. 7,11,12 However, these studies, like flux studies, are complicated by varying numbers of reticulocytes (or young cells) in unfractionated SS blood, which preclude comparisons of KCC-mediated volume reduction in SS and AA cells. We recently compared the RVD mediated by KCC and stimulated by cell swelling and acid pH in SS and AA reticulocytes by tracking the changes in reticulocyte CHC by means of density gradient analysis. 8 Acid-stimulated volume reduction was exaggerated in SS reticulocytes and was diminished by sulfhydryl reduction, parallel to the behavior of KCC flux activity. However, despite the normal response of KCC flux activation to cell swelling in SS RBCs, swelling-stimulated volume reduction was markedly abnormal in SS reticulocytes and resulted in higher final CHC than in AA reticulocytes. Thus, volume reduction and KCC fluxes appear to reflect different functional aspects of the KCC system in RBCs and may be subject to different pathologic influences in SS RBCs...

A novel erythroid anion exchange variant (Gly796Arg) of hereditary stomatocytosis associated with dyserythropoiesis

“…15 In urea-treated red cells, we observed increased tyrosine-phosphorylation of proteins with a molecular weight greater than 181 KDa, one band between 181-115 KDa and one at 82 KDa which were also found in diseased red cells (bands 1, 2, 3; Figure 5A), suggesting a possible adaptive mechanism of red cells to swelling involving these proteins. However, in diseased red cells we observed additional changes in tyrosine phosphorylation state of other membrane proteins, indicating an independent effect of the hematologic phenotype ( Figure 5A).…”

“…To evaluate whether cell swelling induced changes in the tyrosine-phosphorylation profile of red cell membrane proteins, control red cells were incubated with and without urea (600 mmol/L final concentration) as previously described by Joiner et al 15 and red-cell ghosts were prepared for immunoblot analysis with specific antiphosphotyrosine antibodies. In some experiments tyrosine-enriched proteins were obtained by immunoprecipitation with a specific anti-phosphotyrosine antibody (clone 4G10, UpState, NY, USA) as previously reported by De Franceschi et al 16,17 Briefly, red-cell ghosts were solubilized in a medium containing 50 mmol/L Tris-HCl, pH 7.4, 100 mmol/L NaCl, 5 mmol/L EDTA, 1% Triton X-100, 1 mmol/L Na-orthovanadate, 0.004% benzamidine, and 1 tablet of a protease inhibitor cocktail (Roche, Germany).…”

Sites of Regulated Phosphorylation that Control K-Cl Cotransporter Activity

“…Human red blood cells were subjected to hypotonic stress (Joiner et al, 2007). Washed erythrocyte cell pellets were resuspended in either ∼320 mOSM isotonic media or ∼220 mOsm hypotonic media and incubated for 10 min at 37°C.…”

“…Phosphorylation activates NKCC1 and inhibits KCCs, while dephosphorylation has opposite effects (Dunham et al, 1980;Jennings and Schulz, 1991;Altamirano et al, 1988;Lytle and Forbush, 1992;Haas and Forbush, 2000;Adragna et al, 2004) (Figure 1). The modulation of [Cl − ] i in response to osmotic challenge is rapid, occurring within minutes (Lang et al, 1998;Joiner et al, 2007). KCC activation in hypotonic stress is ablated by the phosphatase inhibitor calyculin A, suggesting an essential role of cotransporter phosphorylation in the acute response (Adragna et al, 2004).…”

Disruption of erythroid K-Cl cotransporters alters erythrocyte volume and partially rescues erythrocyte dehydration in SAD mice

“…Erythroid K-Cl cotransport activity is increased in sickle cell disease (SCD) (16) and is thought to contribute to rbc dehydration and the formation of sickle cells (17,18). SCD is generally caused by the hemoglobin (Hb) β-chain E6V mutation.…”