A novel method using confocal laser scanning microscopy for sensitive measurement of P-glycoprotein-mediated transport activity in Caco-2 cells

• Published in: Journal of pharmacy and pharmacology Vol. 63; no. 8; pp. 1015 - 1021
• Main Authors: Wakuda, Hirokazu, Nejime, Namie, Tada, Yukari, Kagota, Satomi, Fahmi, Odette A., Umegaki, Keizo, Yamada, Shizuo, Shinozuka, Kazumasa
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01-08-2011
• Subjects: ATP-Binding Cassette, Sub-Family B, Member 1 - metabolism; Biological Transport - drug effects; Biological Transport - physiology; Caco-2 cells; Caco-2 Cells - metabolism; confocal laser scanning microscopy; digoxin; Digoxin - pharmacology; Drug Interactions - physiology; Fluorescent Dyes - metabolism; Humans; Hypericum; Microscopy, Confocal - methods; P-glycoprotein; Plant Extracts - pharmacology; Rhodamine 123 - metabolism; St John's wort
• ISSN: 0022-3573; 2042-7158
• DOI: 10.1111/j.2042-7158.2011.01294.x

Objectives  The aim of this study was to use time‐lapse confocal laser scanning microscopy to establish a more sensitive and specific method for evaluating P‐glycoprotein activity in Caco‐2 cells.
Methods  The change in the fluorescence of residual rhodamine 123 at the apical and central regions of Caco‐2 cells was measured in the presence of digoxin or St John's wort by using time‐lapse confocal laser scanning microscopy. The data were compared with measurements made using conventional techniques, a fluorescence microplate reader and a fluorescence microscope.
Key findings  The percentage decrease of rhodamine 123 caused by 10 µm digoxin or 0.1 µg/ml St John's wort was significantly larger in the apical region of the Caco‐2 cell than in the central region or in the whole cell. The digoxin‐induced inhibition in the apical region as measured by time‐lapse confocal laser scanning microscopy was greater than that measured in the whole cell by a microplate reader or a fluorescence microscope.
Conclusions  The assay of residual rhodamine 123 in the apical region of Caco‐2 cells by confocal laser scanning microscopy was more sensitive than the conventional methods using a microplate reader or fluorescence microscopy. It will be a valuable screening tool for studying both the inhibition and induction of P‐glycoprotein activity.