We studied the mechanism of the lidocaine-induced shape change in huma
n erythrocytes. Immunohistochemical analysis of erythrocytes using spe
ctrin-specific antibodies revealed aggregation of fluorescence in lido
caine-treated cells, while the fluorescence was distributed diffusely
in untreated cells. The intracellular pH in lidocaine-treated erythroc
ytes was examined by flow cytometry of the cells labeled with 2'-carbo
xyethyl-6',7'-(dihydropyran-2'-one)-5-carb diacethoxymethylester (BCEC
F-AM), and was found to decrease with increasing concentrations of lid
ocaine. Pre-treatment of erythrocytes with acetazolamide, an inhibitor
of carbonic anhydrase, inhibited the lidocaine-induced spectrin aggre
gation and decrease in intracellular pH. When erythrocytes were incuba
ted in medium containing bafilomycin A(1), an inhibitor of V-ATPase, f
ollowed by incubation with lidocaine, the cells changed shape slightly
and the intracellular pH showed a small decrease in comparison with c
ontrol. Spectrin dimers extracted from membranes of normal erythrocyte
s were incubated in buffers of various pHs and analyzed by SDS-PAGE. T
he amounts of spectrin dimers and tetramers decreased, while that of o
ligomers 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.