The transport kinetics of lanthanide species in a single erythrocyte probed by confocal laser scanning microscopy |
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| Authors: | Y Cheng Qihua Huo Jingfen Lu Rongchang Li K Wang |
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| Institution: | (1) National Research Laboratories of Natural and Biomimetic Drugs, Beijing Medical University, Beijing 100083, China e-mail: wangkui@mail.bjmu.edu.cn Tel.: +86-10-62091539 Fax: +86-10-62015584, CN;(2) Analytical Center of Beijing Medical University, Beijing 100083 China, CN |
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| Abstract: | 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 Ln3+, 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 Ln3+ might be attributed to the enhanced permeability of erythrocytes owing to Ln3+ binding. These findings strongly demonstrate the existence of the non-internalization mechanism of Ln species uptake by erythrocytes.
Received: 7 January 1999 / Accepted: 7 May 1999 |
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| Keywords: | Lanthanide Erythrocyte Transport Confocal laser scanning microscopy |
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