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 degrees C (7.41 +/- 1.26 pmoles/h.cm2 vs. 27.40 +/- 8.91 at 37 degrees C). The amount conveyed via the direct pathway was very small (0.89 +/- 0.35 pmoles/h.cm2) and did not diminish at 4 degrees C (1.43 +/- 0.59 pmoles/h.cm2). In the basal-apical direction, HRP transport along the degradative pathway at 37 degrees C was similar to the apical-basal value and was inhibited at 4 degrees C (16.40 +/- 4.05 vs. 2.72 +/- 2.52 pmoles/h.cm2), but along the direct pathway, it was eight times the apical-basal value (8.36 +/- 3.11 pmoles/h.cm2) and was inhibited at 4 degrees C (2.43 +/- 0.78 pmoles/h.cm2). Intact HRP fluxes were not correlated with the electrical 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.
Heyman
M
M
Inserm U. 290, Hôpital Saint-Lazare, Paris, France.
Crain-Denoyelle
A M
AM
Nath
S K
SK
Desjeux
J F
JF
D001692:Biological Transport / Q000187:drug effects; D003106:Colon / Q000378:metabolism*; D004848:Epithelium / Q000378:metabolism / Q000648:ultrastructure; D006735:Horseradish Peroxidase / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008247:Lysosomes / Q000378:metabolism; D008985:Monensin / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism*; D013696:Temperature; D014407:Tumor Cells, Cultured
D011506:Proteins; D008985:Monensin; D006735:Horseradish Peroxidase