APICAL MEMBRANE-PERMEABILITY OF MDCK CELLS

The osmotic water permeability (P-f) and permeability to nonelectrolyt es were determined for the apical membrane of clonal strain Madin-Darb y canine kidney (MDCK) C12 cells cultured as cysts with the apical mem brane facing the surrounding medium. P-f and solute permeabilities wer e calculated from the rate of volume change of cysts by digitizing ima ges at 1-s intervals after instantaneous osmotic challenge. Image meas urement was fully automated with the use of a program that separated t he image of the cyst from the background by using adaptive intensity t hresholding and shape analysis. P-f, calculated by curve fitting to th e volume loss data, averaged 2.4 +/- 0.1 mu m/s and was increased by a ddition of amphotericin B. The energy of activation for P-f was high ( 16.3 kcal/mol), and forskolin (50 mu M) had no effect on P-f. Two popu lations of MDCK cysts were studied: those with two to three cells and those that appeared to be composed of only one cell. The P-f of multic ell cysts was the same as single cell cysts, suggesting that paracellu lar water flow is not significant. Solute permeability was measured us ing paired osmotic challenges (sucrose and test solute) on the same cy st. Urea permeability was not different from zero, whereas the permeab ilities of acetamide and formamide were consistent with their relative oil-water partition coefficients. Our data are similar to values from studies on the permeability properties of vesicles of water-tight epi thelial apical membrane. The combination of the unique model of MDCK a pical-out cysts and fully automated data analysis enabled determinatio n of apical membrane permeability in intact epithelial cells with high precision.