Highly sensitive measurement of P-glycoprotein-mediated transport activity in Caco-2 cells.

We established a highly sensitive method for evaluating P-glycoprotein activity in Caco-2 cells. Using time-lapse confocal laser-scanning microscopy, we measured the change in fluorescence of residual rhodamine 123 in Caco-2 cells. Horizontal fluorescence images of rhodamine 123 in the apical and central parts of these cells were captured for 90 min. A continuous and significant decrease in the fluorescent intensity of rhodamine 123 in the apical part of the Caco-2 cells occurred during the measurement period, while the decrease in the central part was mild. The decrease in rhodamine 123 intensity in the apical part of Caco-2 cells were abolished in the presence of 10 microM digoxin, but the decrease in the central part was not. The decrease in total rhodamine 123 over whole cells was no significant influence of digoxin was observed. This residual rhodamine 123 assay for evaluating P-glycoprotein in the apical part of Caco-2 cells imaged by confocal laser scanning microscopy is more sensitive than conventional methods and will be a valuable screening tool for studying both the inhibition and induction of P-glycoprotein activity and expression. This method may also be useful for predicting P-glycoprotein-mediated alterations in the intestinal absorption of drugs.