Effects of anion transport inhibitors on hemolysis of human erythrocytes under hydrostatic pressure

Effects of anion transport inhibitors on hemolysis of human erythrocytes at 200 mPa were examined. The degree of hemolysis was decreased by treating intact cells with 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS), 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate, or bis(sulfosuccinimidyl)suberate, whereas 4,4'-dinitrostilbene-2,2'-disulfonate and pyridoxal 5'-phosphate (PLP) had little effect on the hemolysis. In contrast, the degree of hypotonic hemolysis increased upon treatment with anion transport inhibitors. From the relationship between the hemolysis at 200 mPa and anion transport, it was found that high-pressure-induced hemolysis was suppressed by the covalent binding of anion transport inhibitors to band 3. This idea was supported by the finding that the hemolysis at 200 mPa of trypsin-treated erythrocytes was suppressed by DIDS. Furthermore, the spectrin content in vesicles which are released from erythrocyte ghosts by dimyristoylphosphatidylcholine decreased upon DIDS labeling of hand 3, but did not change upon PLP labeling. These results suggest that the interaction of the cytoplasmic domain of band 3 with spectrin, perhaps via ankyrin, is tightened by the covalent binding of bulky ligands to the exofacial domain of band 3.