The cytotoxic effect of long-term exposure of renal epithelial cells to ouabain and other cardiotonic steroids (CTS) is mediated by the interaction of these compounds with Na(+),K(+)-ATPase but is independent of the inhibition of Na(+),K(+)-ATPase-mediated ion fluxes. Sustained application of CTS also leads to Na(+),K(+)-ATPase endocytosis and its translocation into the nuclei that might trigger the cell death machinery via the regulation of gene expression. This study examines the role of Na(+),K(+)-ATPase internalization and de novo gene expression in the death of ouabain-treated C7-Madin-Darby canine kidney (MDCK) cells derived from distal tubules of the MDCK. In these cells, 6-h exposure to 3 microM ouabain led to the internalization of approximately 50% of plasmalemmal Na(+),K(+)-ATPase. Prolonged incubation in a K(+)-free medium abolished ouabain-induced Na(+),K(+)-ATPase internalization but did not affect the cytotoxic action of ouabain seen after 18-h incubation. Previously, it was shown that CTS-induced Na(+),K(+)-ATPase internalization is mediated by its interaction with Src within caveolae. Neither caveolae damage by cholesterol depletion with methyl-beta-cyclodextrin nor Src inhibition with 4-amino-5(4-chlorophenyl)-7-(t-butyl)pyrazol[3,4-d]pyridine affected the death of ouabain-treated C7-MDCK cells. Actinomycin D at the 0.1-microg/ml concentration almost completely abolished ribonucleic acid synthesis but did not protect C7-MDCK cells from the cytotoxic action of ouabain. Our results show that neither Na(+),K(+)-ATPase endocytosis nor de novo gene expression contributes to Na(+)(i), K(+)(i)-independent cell death signaling evoked by prolonged exposure to CTS.