Cardiotonic steroid-resistant α1-Na+,K+-ATPase rescues renal epithelial cells from the cytotoxic action of ouabain: evidence for a Nai+,Ki+-independent mechanism

Mechanisms underlying the tissue-specific impact of cardiotonic steroids (CTS) on cell survival and death remain poorly understood. This study examines the role of Na+,K+-ATPase alpha subunits in death of Madin-Darby canine kidney (MDCK) cells evoked by 24-h exposure to ouabain. MDCK cells expressing a variant of the alpha 1 isoform, CTS-sensitive alpha 1S, were stably transfected with a cDNA encoding CTS-resistant alpha 1R-Na+,K+-ATPase, whose expression was confirmed by RT-PCR. In mock-transfected and alpha 1R-cells, maximal inhibition of Rb-86 influx was observed at 10 and 1000 mu M ouabain, respectively, thus confirming high abundance of alpha 1R-Na+,K+-ATPase in these cells. Six-hour treatment of alpha 1R-cells with 1000 mu M ouabain led to the same elevation of the [Na+](i)/[K+](i) ratio that was detected in mock-transfected cells treated with 3 mu M ouabain. However, in contrast to the massive death of mock-transfected cells exposed to 3 mu M ouabain, alpha 1R-cells survived after 24-h incubation with 1000 mu M ouabain. Inversion of the [Na+](i)/[K+](i) ratio evoked by Na+,K+-ATPase inhibition in K+-free medium did not affect survival of alpha 1R-cells but increased their sensitivity to ouabain. Our results show that the alpha 1R subunit rescues MDCK cells from the cytotoxic action of CTS independently of inhibition of Na+,K+-ATPase-mediated Na+ and K+ fluxes and inversion of the [Na+](i)/[K+](i) ratio.