The transport kinetics of lanthanide species in a single erythrocyte probed by confocal laser scanning microscopy

A novel method has been developed to visualize and follow the temporal course of lanthanide transport across the membrane into a single living erythrocyte. By means of confocal scanning microscopy and the optical section technique, the entry of lanthanide ions was followed by the fluorescence quenching of fluorescein isothiocyanate (FITC)-labeled membrane and cytosol. From the difference of the quenching kinetics of the whole section and the central area, the time for diffusion through the membrane and the diffusion in the extracellular and intracellular media can be deduced. To clarify the mechanism of lanthanide-induced fluorescence quenching of FITC-labeled erythrocytes and to ensure that this reaction can be used in this method, the reaction was investigated by steady-state fluorescence techniques. The results showed that the lanthanides strongly quenched the florescence emitted by FITC covalently bound to membrane proteins and cytosolic proteins. The static quenching mechanism is responsible for the fluorescence quenching of FITC-labeled proteins by Ln species. The quenching mechanism is discussed on the basis of complex formation. The dependence of fluorescence quenching on both ion size and the total orbital angular momentum L supports the complexation mechanism. The transport time across the membrane is strikingly correlated with Ln species and extracellular concentration. For a given concentration, the transport time of [Ln(cit)(2)](3-) is much shorter than that of Ln(3+), since they enter the cells via the anion channel. This is supported by the inhibition effect of 4,4'-diisothiocyanato-2,2'-stilbenendisulfonate on the transport of [Ln(cit)(2)](3-). On the other hand, the transport of free Ln(3+) might be attributed to the enhanced permeability of erythrocytes owing to Ln(3+) binding. These findings strongly demonstrate the existence of the non-internalization mechanism of Ln species uptake by erythrocytes.