Permeabilization of MDCK cells with cholesterol binding agents: dependence on substratum and confluency

We studied systematically the susceptibility of Madin-Darby canine kidney (MDCK) cells to permeabilization by two cholesterol binding agents, digitonin and streptolysin-O (SLO), under different culture conditions. Monolayers grown on polycarbonate filter chambers (Transwells) required twice the concentration of digitonin effective on monolayers grown on glass or plastic (80 vs. 40 micrograms/ml) to allow antibody penetration or the release of 90% of the cytosolic protein lactate dehydrogenase (LDH). Neither the apical nor the basolateral surface showed preferential susceptibility to digitonin. Confluent MDCK cells, cultured either on filters or on impermeable substrates, showed poor antibody permeability after addition of commercial SLOs, even when used at concentrations 100 times higher (20 U/ml) than those effective on nonepithelial Chinese hamster ovary cells. Surprisingly, culture conditions that prevent tight junction formation and the acquisition of a polarized phenotype (< 10 microM Ca2+) increased dramatically the susceptibility to permeabilization by SLO. On restoration of normal Ca2+ levels, susceptibility to SLO quickly decreased. Thus conditions that lead to the full establishment of polarity result in decreased sensitivity to disruption by digitonin and SLO.