Figure 3. Estrogen receptor inhibitor ICI 182,780 (ICI) prevented estradiol 17ß-d-glucuronide (E17G)-induced endocytic internalization of Abcb11 and Abcc2 in IRHC.

Panel A shows representative confocal images depicting cellular distribution of the canalicular transporters studied under the different treatments. Note that under control or ICI conditions transporter-associated fluorescence is mainly localized at the canalicular membrane. E17G (200 µM) induced a clear internalization of transporter-containing vesicles beyond the limits of the canaliculus, phenomenon significantly prevented by pretreatment with ICI (1 µM, 15 min). Panel B shows the densitometric analysis of the distribution of Abcb11 and Abcc2 fluorescence intensity along an 8-µm line perpendicular to the canalicular vacuole (4 µm to each side of the vacuole center), using the ImageJ 1.44p software (NIH, USA). The canalicular space was identified based on the corresponding DIC image. Each line profile measurement was normalized to the sum of all intensities of the respective measurement. The distribution of Abcb11 or Abcc2 fluorescence, expressed as a percentage of the total, was then calculated for each canaliculus and compared statistically, using the Mann-Whitney test. Statistical analysis of the profiles revealed a significant internalization of Abcb11 and Abcc2 under E17G treatment (p<0.05 vs control), which was completely abolished by ICI (p<0.05 vs E17G). Results are expressed as mean ± SEM. n = 6–8 canalicular vacuoles per preparation, from 3 independent preparations.