Effects of intracellular pH on high pressure-induced hemolysis of anion transport inhibitor-treated erythrocytes

Effects of anion transport inhibitors such as 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate on hemolysis of human erythrocytes at 200 MPa were examined by changing intracellular pH (7.2-7.9). These inhibitors suppressed the hemolysis at neutral pH but enhanced it at alkaline pH. However, such an enhancement was suppressed by cross-linking of membrane proteins using diamide. From the near-UV CD spectra of band 3 and the relation between hemolysis and anion transport in intact or trypsin-treated erythrocytes, it was found that such hemolytic properties were characterized by the binding of inhibitors to band 3. In addition, spectrin detachment from the erythrocyte membrane by high pressure was considerably suppressed by DIDS treatment at neutral pH, but not by DIDS labeling at alkaline pH. These results suggest that the interaction of the cytoplasmic domain of band 3 with the cytoskeleton, which is induced by the binding of ligands to the exofacial domain of band 3, is dependent on the intracellular pH, i.e., the linking is tightened at neutral pH but relaxed at alkaline pH.