Ouabain resistance of the epithelial cell line (Ma104) is not due to lack of affinity of its pumps for the drug

Na+, K(+)-pumps of most eukaryotic animal cells bind ouabain with high affinity, stop pumping, and consequently loose K+, detach from each other and from the substrate, and die. Lack of affinity for the drug results in ouabain resistance. In this work, we report that Ma104 cells (epithelial from Rhesus monkey kidney) have a novel form of ouabain-resistance: they bind the drug with high affinity (Km about 4 x 10(-8) M), they loose their K+ and stop proliferating but, in spite of these, up to 100% of the cells remain attached in 1.0 microM ouabain, and 53% in 1.0 mM. When 4 days later ouabain is removed from the culture medium, cells regain K+ and resume proliferation. Strophanthidin, a drug that attaches less firmly than ouabain, produces a similar phenomenon, but allows a considerably faster recovery. This reversal may be associated to the fact that, while in ouabain-sensitive MDCK cells Na+, K(+)-ATPases blocked by the drug are retrieved from the plasma membrane, those in Ma104 cells remain at the cell-cell border, as if they were cell-cell attaching molecules. Cycloheximide (10 micrograms/ml) and chloroquine (10 microM) impair this recovery, suggesting that it also depends on the synthesis and insertion of a crucial protein component, that may be different from the pump itself. Therefore ouabain resistance of Ma104 cells is not due to a lack of affinity for the drug, but to a failure of its Na+, K(+)-ATPases to detach from the plasma membrane in spite of being blocked by ouabain.