Erythrocyte (RBC) dehydration is seen in several hematological disorders, including sickle cell anemia, thalassemias, and hereditary spherocytosis (HS). Murine models of HS exhibit increased cell Na and reduced K contents, and increased passive permeability to monovalent cations, like the human counterpart. Mice with complete deficiency of all 4.1R protein isoforms (4.1−/−) exhibit moderate hemolytic anemia, with spherocytosis, and decreased membrane stability (

Shi TS et al., J. Clin. Invest. 103:331,1999
). We examined the effect of 4.1R deficiency on RBC ion transport and volume regulation: 4.1−/− RBCs exhibit cell dehydration, with reduced cell K content (392.3±38.4 vs. 444.2±14.4 mmol/Kg/Hb), associated with up-regulated K-Cl cotransport and Gardos channel activities. 4.1−/− RBCs exhibit markedly increased Na content (48.5±8.8 vs. 13±2.0 mmol/Kg/Hb) and Na permeability, the latter mostly mediated by Na/H exchange (NHE1). The Na/H exchange of 4.1−/− RBCs is markedly activated by exposure to hypertonic conditions, (Vmax 18.2±3.2 vs. 9.8±1.3 mmol/1013cellxh) with an abnormal dependence on osmolarity (K50 of 417±42 vs 460±35 mOsm), suggestive of an up-regulated functional state. PKC inhibition (with either Chelerythrine or calphostin C) did not affect the volume-sensitive Na/H exchange activity in either control or 4.1 −/− RBCs. The protein phosphatase inhibitors okadaic acid (OKA) and calyculin A (CA) significantly stimulated the Na/H exchanger activity in control RBCs while they paradoxically inhibited it in 4.1−/− RBCs. In the presence of OKA, a complete loss of the Na/H exchange activation by hypertonic shrinkage was observed in 4.1 −/− RBCs. In 4.1 −/− RBCs, the Vmax of the Gardos channel was found to be significantly higher (from 6.08 to 9.75 ± 1.06 mmol/L cell x min, n=3, p<0.04) and the affinity constant for Ca2+to be significantly lower (from 1.47 to 1.01 ± 0.06 mM, n=3, p<0.03) than in control. When 4.1 −/− mice were treated with oral clotrimazole (CLT; 80 mg/Kg/BID for 11 days or 160 mg CLT/kg/BID for 48 hrs), a well-characterized Gardos channel blocker, worsening of anemia, increased mortality and increased cell dehydration/fragmentation were noted. Essentially qualitatively similar results were obtained with o-Chlorophenyl-diphenyl acetamide, a CLT analog devoid of the imidazole moiety in 4.1 −/− cells, and with CLT in 4.2−/−, and band 3 −/+ mice (160 mg/kg/BID for 6 and 2 days, respectively), indicating that K and water loss via the Gardos channel may play an important protective role in compensating for the reduced surface membrane area of HS RBCs. In conclusion, 4.1 −/− RBCs exhibit distinct functional abnormalities, indicative of an important functional interaction between protein 4.1R and NHE1. In addition, the use of Gardos channel inhibitors in vivo is associated with worsening anemia, hemolysis and red cell fragmentation, indicating that this pathway may play an important role in protecting spherocytes from reaching their critical hemolytic volume.

Topics:
hemolysis, mice, spherocytosis, dehydration, anemia, acetamides, clotrimazole, hematological diseases, hemolytic anemia, hereditary spherocytosis

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2005, The American Society of Hematology
2004
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