Intracellular turnover of fluorescein diacetate. Influence of membrane ionic gradients on fluorescein efflux.

The influence of the membrane ionic gradient on the efflux of Fluorescein after intracellular turnover of Fluorescein diacetate was studied in HeLa cells. The kinetics of Fluorescein efflux was monitored by determining with flow cytometry the decrease in fluorescence intensity of single cells. Alterations of the Na+ and K+ gradients were induced experimentally by using ouabain, ionophores or buffers in which the ion concentration was modified. The sodium gradient was also altered by using Na(+)-co-transported amino acids. Independent evidence of these changes was obtained with a potential-sensitive indicator, 3,3'-dihexyloxacarbocyanine iodide. Conditions inducing a reduction or dissipation of the ionic gradients caused a decrease in the rate constant of Fluorescein release. In contrast, enhancement of the gradients increased the efflux rate. These results indicate that the release of Fluorescein from living cells is influenced by the membrane potential. Thus, the turnover of Fluorescein diacetate may provide a useful technique for assessing changes in membrane permeability properties related to ionic gradients.