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 shown in earlier studies to induce dose-dependent changes in red blood cell (RBC) shape and volume at the same low concentrations that inhibit anion transport. In the present work, these ligand-induced morphology and rheology changes were studied using video digital image morphometry (VDIM) and microfiltration techniques. The results of these experiments corroborate our earlier investigation. RBCs were filmed using a Nomarski optics microscope with video camera attachment and cell size and shape changes were computer analyzed using VDIM. Low microM p-AzBPhz or DIDS levels caused collapse of the cell's biconcave structure and cell flattening occurred within 1-2 sec after drug exposure. Higher doses of either agent converted cells to a new steady-state in which a concurrent limited increase in erythrocyte volume and blunt membrane protrusions were produced. These changes were reversed in less than 2 sec by washing the drug from the membrane. Both ligands increased the deformability of RBCs in a dose-dependent manner as determined by filtration through Nuclepore polycarbonate filters (3 microns pore diameter). The improvement in deformability of drug-treated sickle cells was much more dramatic than for normal cells at low p-AzBPhz concentrations. These 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.
Center of Membrane Sciences, University of Kentucky College of Medicine, Lexington 40506-0057.
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Hoefner
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Blank
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BM
Davis
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Diedrich
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology*; D001386:Azides / Q000494:pharmacology*; D048429:Cell Size / Q000187:drug effects*; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008855:Microscopy, Electron, Scanning; D010695:Phlorhizin / Q000031:analogs & derivatives* / Q000494:pharmacology
D001386:Azides; C056185:phlorizinyl 5'-benzylazide; D010695:Phlorhizin; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid