Abstract
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'-aeetyl-2'-carboxyethyl-6', 7'-(dihydropyran-2'-one)-5-carboxyfluorescein 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 membranesof 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.
