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Permeability of human HT-29/B6 colonic epithelium as a function of apoptosis

C. Bojarski *, A. H. Gitter †, K. Bendfeldt †, J. Mankertz *, H. Schmitz *, S. Wagner ‡, M. Fromm † and J. D. Schulzke *

1. The barrier function of colonic epithelia is challenged by apoptotic loss of enterocytes. In monolayers of human colonic HT-29/B6 cells, apoptosis induced by camptothecin was assessed by poly-(ADP-ribose)-polymerase (PARP) cleavage, histone ELISA and DNA-specific fluorochrome staining (with 4',6'-diamidino-2'-phenylindoladihydrochloride (DAPI)). Epithelial barrier function was studied in Ussing chambers by measuring transepithelial conductivity and unidirectional tracer fluxes. The ion permeability associated with single cell apoptoses was investigated with the conductance scanning technique.
2. The spontaneous rate of apoptotic cells was 3.5 ± 0.3 % with an overall epithelial conductivity of 3.2 ± 0.1 mS cm^-2. Camptothecin induced a time- and dose-dependent increase of apoptosis and permeability. With 20 µg ml^-1 of camptothecin for 48 h, apoptosis increased 4.1-fold to 14.3 ± 1.5 % and the conductivity doubled to 6.4 ± 1.0 mS cm^-2.
3. While ³H-mannitol flux increased 3.8-fold and ³H-lactulose flux increased 2.6-fold, the flux of ³H-polyethylene glycol 4000 remained unchanged. Hence, the higher permeability was limited to molecules < 4000 Da.
4. The local epithelial conductivity was higher at the sites of apoptosis than in non-apoptotic areas. With camptothecin the leaks associated with apoptosis became more numerous and more conductive, while in non-apoptotic areas the conductivity remained at control level. Hence, the camptothecin-induced increase in epithelial conductivity reflected the opening of apoptotic leaks and thus the results described, for the first time, epithelial permeability as a function of apoptosis only.
5. The conductivity of apoptotic leaks contributed 5.5 % to the epithelial conductivity of controls and 60 % to the conductivity of monolayers treated with 20 µg ml^-1 of camptothecin. Thus apoptosis increased the contribution of paracellular pathways to the overall epithelial permeability. Under control conditions the paracellular conductivity (G_para) was smaller than the transcellular (G_trans), but with 12 % apoptosis, G_para exceeded G_trans. By definition, the epithelium became 'leaky'.

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