Nishiguchi E, Ozono S, Takakuwa Y, Hamasaki N
Department of Dental Hygiene, Shonan Junior College, Yokosuka, Japan.
Cell Struct Funct. 1995 Feb;20(1):71-9. doi: 10.1247/csf.20.71.
We studied the mechanism of the lidocaine-induced shape change in human erythrocytes. Immunohistochemical analysis of erythrocytes using spectrin-specific antibodies revealed aggregation of fluorescence in lidocaine-treated cells, while the fluorescence was distributed diffusely in untreated cells. The intracellular pH in lidocaine-treated erythrocytes was examined by flow cytometry of the cells labeled with 3'-acetyl-2'-carboxy-ethyl-6',7'-(dihydropyran-2'-one)-5-carboxyfluoresc ein diacethoxymethylester (BCECF-AM), and was found to decrease with increasing concentrations of lidocaine. Pre-treatment of erythrocytes with acetazolamide, an inhibitor of carbonic anhydrase, inhibited the lidocaine-induced spectrin aggregation and decrease in intracellular pH. When erythrocytes were incubated in medium containing bafilomycin A1, an inhibitor of V-ATPase, followed by incubation with lidocaine, the cells changed shape slightly and the intracellular pH showed a small decrease in comparison with control. Spectrin dimers extracted from membranes normal erythrocytes were incubated in buffers of various pHs and analyzed by SDS-PAGE. The amounts of spectrin dimers and tetramers decreased, while that of oligomers increased with decreasing pH. These results suggest that the lidocaine-induced shape change in human erythrocytes may occur by the conformational change of spectrin in a process that may be mediated by carbonic anhydrase and activation of V-ATPase.
我们研究了利多卡因诱导人红细胞形状改变的机制。使用血影蛋白特异性抗体对红细胞进行免疫组织化学分析显示，利多卡因处理的细胞中荧光聚集，而未处理的细胞中荧光呈弥散分布。通过对用3'-乙酰-2'-羧基-乙基-6',7'-(二氢吡喃-2'-酮)-5-羧基荧光素二乙酰氧基甲酯（BCECF-AM）标记的细胞进行流式细胞术检测利多卡因处理的红细胞内pH值，发现其随着利多卡因浓度的增加而降低。用碳酸酐酶抑制剂乙酰唑胺预处理红细胞，可抑制利多卡因诱导的血影蛋白聚集和细胞内pH值降低。当红细胞在含有V-ATP酶抑制剂巴弗洛霉素A1的培养基中孵育，然后再与利多卡因孵育时，细胞形状略有改变，与对照相比细胞内pH值略有下降。从正常红细胞膜中提取的血影蛋白二聚体在不同pH值的缓冲液中孵育，并通过SDS-PAGE进行分析。随着pH值降低，血影蛋白二聚体和四聚体的量减少，而寡聚体的量增加。这些结果表明，利多卡因诱导的人红细胞形状改变可能是通过血影蛋白的构象变化发生的，这一过程可能由碳酸酐酶和V-ATP酶的激活介导。
