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

p-Azidobenzylphlorizin (p-AzBPhz) is a potential photoaffinity labelin g agent for the anion and glucose transporters in human RBCs. In the a bsence of light and at the same low concentrations which block these t ransport processes (only 1-2 million molecules bound/cell), this imper meable membrane probe produces rapid morphological and volume alterati ons. This high-affinity activity, called phase 1, can be rapidly and c ompletely reversed by simply diluting the azide-treated cell suspensio n. Phase 2 effects, including formation of cells with multiple, long s picules (stage 3/4 echinocytes), followed by an influx of salt and wat er 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 cel l sizing, microhematocrit measurements and scanning electron microscop y have been employed to compare the effects of the azide and the anion transport inhibitor, DIDS (4,4'-diisothiocyano-2,2'-stilbene disulfon ate), on red cells. DIDS produced only those changes analogous to the azide's low dose phase 1 action; cells swell, lose the ability to scat ter 800 nm light and undergo a limited shape change (comparable to sta ge 1 echinocytosis). The mechanism by which the two ligands perturb th e membrane is additive, suggesting that a Band 3-mediated transmembran e signaling is involved which leads to altered cytoskeleton dynamics.