Hoefner D M, Blank M E, Davis B M, Diedrich D F
Center of Membrane Sciences, University of Kentucky College of Medicine, Lexington 40506-0057.
J Membr Biol. 1994 Jul;141(1):91-100. doi: 10.1007/BF00232877.
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.
两种非穿透性膜探针，对叠氮苄基根皮苷（p-AzBPhz）和4,4'-二异硫氰基-2,2'-二苯乙烯二磺酸（DIDS），在早期研究中已表明，在抑制阴离子转运的相同低浓度下，可诱导红细胞（RBC）形状和体积发生剂量依赖性变化。在本研究中，使用视频数字图像形态测量法（VDIM）和微滤技术研究了这些配体诱导的形态和流变学变化。这些实验结果证实了我们早期的研究。使用配备摄像机的诺马斯基光学显微镜拍摄红细胞，并使用VDIM对细胞大小和形状变化进行计算机分析。低 microM浓度的p-AzBPhz或DIDS会导致细胞双凹结构塌陷，药物暴露后1-2秒内细胞会变平。更高剂量的任何一种试剂都会使细胞转变为新的稳态，其中红细胞体积会同时有限增加，并产生钝性膜突起。通过从膜上洗去药物，这些变化在不到2秒内就会逆转。通过Nuclepore聚碳酸酯滤膜（孔径3微米）过滤测定，两种配体均以剂量依赖性方式增加了红细胞的变形性。在低p-AzBPhz浓度下，药物处理的镰状细胞变形性的改善比正常细胞更为显著。这些结果支持了我们早期的结论，即配体通过与带3的共同相互作用，诱导与体积相关的细胞骨架改变，从而导致形态和柔韧性的变化。
