title: A novel method using confocal laser scanning microscopy for sensitive measurement of P-glycoprotein-mediated transport activity in Caco-2 cells.
abstract: OBJECTIVE
  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.
  
  
  RESULTS
  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.
authors: Department of Pharmacology, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women's University, Nishinomiya, Japan.
authors: Hirokazu
authors: H
authors: Wakuda
authors: Namie
authors: N
authors: Nejime
authors: Yukari
authors: Y
authors: Tada
authors: Satomi
authors: S
authors: Kagota
authors: Odette A
authors: OA
authors: Fahmi
authors: Keizo
authors: K
authors: Umegaki
authors: Shizuo
authors: S
authors: Yamada
authors: Kazumasa
authors: K
authors: Shinozuka
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology*; D018938:Caco-2 Cells / Q000378:metabolism*; D004077:Digoxin / Q000494:pharmacology; D004347:Drug Interactions / Q000502:physiology*; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D020902:Hypericum; D018613:Microscopy, Confocal / Q000379:methods*; D010936:Plant Extracts / Q000494:pharmacology; D020112:Rhodamine 123 / Q000378:metabolism
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D005456:Fluorescent Dyes; D010936:Plant Extracts; D020112:Rhodamine 123; D004077:Digoxin