Quantification of protein transcytosis in the human colon carcinoma cell line CaCo‐2

The transepithelial absorption of food‐type proteins has been shown to proceed by endocytosis along two functional pathways: a minor direct pathway allowing transport of intact protein and a major lysosomal degradative pathway. The human colon carcinoma cell line CaCo‐2 grown on Millipore filters was used here further to characterize these pathways by measuring HRP transport across the cell monolayer in Ussing chambers. In the apical‐basal direction, this transport mainly occurred along the degradative pathway and was inhibited at 4°C (7.41 ± 1.26 pmoles/h·cm 2 vs. 27.40 ± 8.91 at 37°C). The amount conveyed via the direct pathway was very small (0.89 ± 0.35 pmoles/h·cm 2 ) and did not diminish at 4°C (1.43 ± 0.59 pmoles/h·cm 2 ). In the basal‐apical direction, HRP transport along the degradative pathway at 37°C was similar to the apical‐basal value and was inhibited at 4°C (16.40 ± 4.50 vs. 2.72 ± 2.52 pmoles/h·cm 2 ), but along the direct pathway, it was eight times the apical‐basal value (8.36 ± 3.11 pmoles/h·cm 2 ) and was inhibited at 4°C (2.43 ± 0.78 pmoles/h·cm 2 ). Intact HRP fluxes were not correlated with the eletrical resistance of the filters, indicating transport via a transcellular route. Monensin at 10 −5 M did not affect direct or degradative transport in the apical‐to‐basal direction. These results suggest that in CaCo‐2 cells HRP undergoes bidirectional transcytosis by a fluid‐phase mechanism, but the extent of degradation during that transport varies according to the membrane (apical or basal) where it is presented.