MORPHOLOGY AND VOLUME ALTERATIONS OF HUMAN ERYTHROCYTES CAUSED BY THE ANION TRANSPORTER INHIBITORS, DIDS AND P-AZIDOBENZYLPHLORIZIN

p-Azidobenzylphlorizin (p-AzBPhz) is a potential photoaffinity labeling agent for the anion and glucose transporters in human RBCs. In the absence of light and at the same low concentrations which block these transport processes (only 1-2 million molecules bound/cell), this impermeable membrane probe produces rapid morphological and volume alterations. This high-affinity activity, called phase 1, can be rapidly and completely reversed by simply diluting the azide-treated cell suspension. Phase 2 effects, including formation of cells with multiple, long spicules (stage 3/4 echinocytes), followed by an influx of salt and water with eventual lysis, occur at two log units higher concentration by a different mechanism, probably by intercalating into and selectively expanding the outer lipid monolayer. Light scattering, electronic cell sizing, microhematocrit measurements and scanning electron microscopy have been employed to compare the effects of the azide and the anion transport inhibitor, DIDS (4,4'-diisothiocyano-2,2'-stilbene disulfonate), on red cells. DIDS produced only those changes analogous to the azide's low dose phase 1 action; cells swell, lose the ability to scatter 800 nm light and undergo a limited shape change (comparable to stage 1 echinocytosis). The mechanism by which the two ligands perturb the membrane is additive, suggesting that a Band 3-mediated transmembrane signaling is involved which leads to altered cytoskeleton dynamics.