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

1. The barrier function of colonic epithelia is challenged by apoptotic los s 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 func tion was studied in Ussing chambers by measuring transepithelial conductivi ty and unidirectional tracer fluxes. The ion permeability associated with s ingle cell apoptoses was investigated with the conductance scanning techniq ue. 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 t ime- and dose-dependent increase of apoptosis and permeability. With 20 mug 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. H-3-mannitol flux increased 3.8-fold and H-3-lactulose flux incre ased 2.6-fold, the flux of H-3-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 t han in non-apoptotic areas. With camptothecin the leaks associated with apo ptosis became more numerous and more conductive, while in non-apoptotic are as the conductivity remained at control level. Hence, the camptothecin-indu ced increase in epithelial conductivity reflected the opening of apoptotic leaks and thus the results described, for the first time, epithelial permea bility as a function of apoptosis only. 5. The conductivity of apoptotic leaks contributed 5.5% to the epithelial c onductivity of controls and 60% to the conductivity of monolayers treated w ith 20 <mu>g ml(-1) of camptothecin. Thus apoptosis increased the contribut ion 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(t rans). By definition, the epithelium became 'leaky'.