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.

The anion transporter and a 28 kDa protein are selectively photolabeled by p-azidobenzylphlorizin under conditions that alter RBC morphology, flexibility, and volume.

Tritiated p-azidobenzylphlorizin (p-AzBPhz) was photoactivated in the presence of red blood cells under conditions previously found to alter morphology, flexibility and volume. When less than 0.25 million molecules were added per cell, only a 28 kDa peptide was photolabeled: at 1-2 million molecules added, band 3 also incorporated significant radioactivity. When using leaky ghosts, other proteins became labeled, including those limited to the cytoplasm. Protein N-deglycosylation caused a shift of radiolabeled band 3 to higher Rf values on SDS-PAGE gels but not for the 28 kDa band; the latter was, however, susceptible to enzymatic digestion by NANase (N-acetylneuraminidase) III but not by NANase II. Inhibition of photoincorporation into both receptors by unlabeled p-AzBPhz was dose-dependent. Mercuric chloride and p-CMBS selectively blocked 28 kDa peptide labeling. DIDS partially blocked at band 3; after 15% inhibition, greater DIDS concentrations caused increased incorporation into the 28 kDa peptide. These results, and a temperature-dependent labeling pattern, suggest that: (i) cellular changes occur when p-AzBPhz binds to the exofacial sides of the anion transporter and 28 kDa peptide; (ii) these proteins may be physically associated in the native membrane; (iii) they mediate ligand-induced changes in morphology, flexibility, and volume.

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.

Development of a rapid, quantitative method for LDL subfractionation with use of the Quantimetrix Lipoprint LDL System.

BACKGROUND: Recent evidence suggests that the presence of small, dense LDL is independently associated with increased risk of developing coronary artery disease. Current methods to subfractionate LDL are time-consuming and/or technically demanding. Therefore, we have sought the development of a less complex LDL subfractionation procedure. METHODS: LDL subfractions were separated using the Quantimetrix Lipoprint(TM) LDL System. High-resolution 3% polyacrylamide gel tubes were scanned densitometrically (610 nm) with a Helena EDC system. A computerized method to identify and quantitatively score the resolved LDL subfractions was developed. Results from the Quantimetrix method were compared using 51 plasma samples with values obtained by nondenaturing gradient gel electrophoresis (NDGGE) and nuclear magnetic resonance (NMR) spectroscopy. RESULTS: LDL subfractionation scores correlated significantly (P <0.05) with triglyceride, HDL-cholesterol, apolipoprotein B100, and LDL-cholesterol/apolipoprotein B100 (r = 0.591, -0.392, 0.454, and -0.411, respectively). For 51 samples, the Quantimetrix method classified 21 with small, 14 with intermediate, and 16 with large LDL. Of the 21 samples classified as small by Quantimetrix, 20 (95%) were classified as small (n = 18) or intermediate (n = 2) by NDGGE. All of the 16 specimens classified as large by Quantimetrix were either large (n = 14) or intermediate (n = 2) by NDGGE. LDL score was inversely correlated (r = -0.674; P <0.0001) with LDL particle size determined by NMR spectroscopy. CONCLUSIONS: A quantitative method for the assessment of LDL particle size phenotype was developed using the Quantimetrix Lipoprint LDL System. The method can be performed in less than 3 h in batch mode and is suitable for routine use in clinical laboratories.