cytochalasin-b and Anemia--Sickle-Cell

Abstract  Red blood cells (RBCs) from patients with sickle cell disease (SCD) lyse in deoxygenated isosmotic non-electrolyte solutions. Haemolysis has features which suggest that it is linked to activation of the pathway termed Psickle. This pathway is usually described as a non-specific cationic conductance activated by deoxygenation, HbS polymerisation and RBC sickling. The current work addresses the hypothesis that this haemolysis will provide a novel diagnostic and prognostic test for SCD, dependent on the altered properties of the RBC membrane resulting from HbS polymerisation. A simple test represented by this haemolysis assay would be useful especially in less affluent deprived areas of the world where SCD is most prevalent. RBCs from HbSS and most HbSC individuals showed progressive lysis in deoxygenated isosmotic sucrose solution at pH 7.4 to a level greater than that observed with RBCs from HbAS or HbAA individuals. Cytochalasin B prevented haemolysis. Haemolysis was temperature- and pH-dependent. It required near physiological temperatures to occur in deoxygenated sucrose solutions at pH 7.4. At pH 6, haemolysis occurred even in oxygenated samples. Haemolysis was reduced in patients on long-term (>5 months) hydroxyurea treatment. Several manoeuvres which stabilise soluble HbS (aromatic aldehydes o-vanillin or 5-hydroxymethyl, and urea) reduced haemolysis, an effect not due to increased oxygen affinity. Conditions designed to elicit HbS polymerisation in cells from sickle trait patients (deoxygenated hyperosmotic sucrose solutions at pH 6) supported their haemolysis. These findings are consistent with haemolysis requiring HbS polymerisation and support the hypothesis that this may be used as a test for SCD.

Topics: Aldehydes; Anemia, Sickle Cell; Cell Membrane; Cytochalasin B; Erythrocytes, Abnormal; Hematologic Tests; Hemoglobins; Hemolysis; Humans; Hydrogen-Ion Concentration; Polymerization; Prognosis; Sucrose; Temperature; Urea

This study investigated the effect of acute deoxygenation on membrane permeability characteristics of sickle cells. Measured fluxes of Na+ and K+ in ouabain-inhibited cells, of chloride and sulfate exchange in 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS)-inhibited and untreated cells, and of erythritol, mannitol, and arabinose in cytochalasin B-inhibited cells indicated that a deoxygenation-induced permeability change occurred in sickle cells only for cations and chloride. Monovalent cation permeabilities increased five-fold, and chloride influx into DIDS treated cells was enhanced nearly threefold on sickle cell deoxygenation. In contrast, no detectable increase in permeability to the other solutes was found. To gain perspective on these findings, similar measurements were performed in normal cells treated with diamide, an agent shown by others to induce a coupled increase in membrane permeability and phospholipid translocation, reminiscent of deoxygenation-induced changes in sickle cells. Although the increase in cation permeability was no greater than that in sickled cells, treatment with 2 mmol/L diamide also produced a twofold increase in the first order rate constants for sulfate exchange and mannitol efflux, indicating a relatively nonselective permeability increase that permitted flux of larger solutes than in the case of deoxygenated sickle cells. These results suggest that the deoxygenation of sickle cells induces a permeability increase that is relatively insensitive to charge, but is restrictive with respect to solute size.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anemia, Sickle Cell; Arabinose; Cell Membrane Permeability; Chlorides; Cytochalasin B; Diamide; Dose-Response Relationship, Drug; Erythritol; Erythrocyte Membrane; Humans; Mannitol; Ouabain; Oxidation-Reduction; Potassium; Sodium; Sulfates