Figure 3.

Ontogeny of OSTα-OSTβ mRNA expression and TC transport in Caco-2 cells. Caco-2 cells were seeded at high density onto transwell filters and maintained in DMEM plus 10% fetal calf serum. Two days prior to assaying bile acid transport at 7, 14, 21, and 28 days post-confluence, the cells were switched to DMEM plus 0.5% charcoal-stripped fetal calf serum to remove endogenous bile acids. At the indicated times, the cells were washed and incubated at 37°C for 30 min with 10 μM [³H]taurocholate in a Hanks balanced salt solution with sodium (137 mM) or without sodium (137 mM potassium) added to the apical chamber [32]. The transport is plotted as the pmoles of taurocholate transported corrected for the background transcellular transport measured in the Hanks balanced salt solution lacking sodium. The cells were harvested for RNA isolation to determine the mRNA expression for ASBT, OSTα and OSTβ by real time PCR. The C_T values are the means of triplicate determinations and expression was normalized for GAPDH expression, which did not change between days 7 and 28 post-confluence. The results are plotted relative to the expression at day 7. The media from the basolateral chamber was collected to determine the transcellular transport of taurocholate. TC transport correlated with the appearance of ASBT mRNA at day 14 and increased ~30-fold between days 7 and 28. ASBT mRNA expression increased ~45-fold between days 7 and 28 post-confluence. OSTα and OSTβ mRNA are present at day 7 and increase ~2-fold between days 7 and 28 post-confluence. The C_T values on day 28 were 22.15, 22.44, and 21.91 for ASBT, OSTα, and OSTβ mRNA, respectively. Inset, the mRNA expression is shown relative to the day 7 measurements.