BAND-3 ANTAGONISTS, P-AZIDOBENZYLPHLORIZIN AND DIDS, MEDIATE ERYTHROCYTE SHAPE AND FLEXIBILITY CHANGES AS CHARACTERIZED BY DIGITAL IMAGE MORPHOMETRY AND MICROFILTRATION

Two nonpenetrating membrane probes, p-azidobenzylphlorizin (p-AzBPhz) and 4,4'-diisothiocyano-2,2'-stilbene disulfonate (DIDS), have been sh own in earlier studies to induce dose-dependent changes in red blood c ell (RBC) shape and volume at the same low concentrations that inhibit anion transport. In the present work, these ligand-induced morphology and theology changes were studied using video digital image morphomet ry (VDIM) and microfiltration techniques. The results of these experim ents corroborate our earlier investigation. RBCs were filmed using a N omarski optics microscope with video camera attachment and cell size a nd shape changes were computer analyzed using VDIM. Low mu M p-AzBPhz or DIDS levels caused collapse of the cell's biconcave structure and c ell flattening occurred within 1-2 sec after drug exposure. Higher dos es of either agent converted cells to a new steady-state in which a co ncurrent limited increase in erythrocyte volume and blunt membrane pro trusions were produced. These changes were reversed in less than 2 sec by washing the drug from the membrane. Both ligands increased the def ormability of RBCs in a dose-dependent manner as determined by filtrat ion through Nuclepore polycarbonate filters (3 mu m pore diameter). Th e improvement in deformability of drug-treated sickle cells was much m ore dramatic than for normal cells at low p-AzBPhz concentrations. The se results support our earlier conclusions that the ligands, through a common interaction with band 3, induce volume-associated cytoskeletal alterations which lead to changes in morphology and flexibility.