Modest intracellular acidification suppresses death signaling in ouabain-treated cells

The signaling cascade resulting in the death of several types of cells treated with ouabain or other cardiotonic steroids (CTS) remains poorly understood. Recently, we observed that ouabain kills epithelial and endothelial cells via its interaction with Na+, K+-ATPase, but independently of inhibition of Na+, K+ ATPase- mediated ion fluxes and inversion of the [Na+](i)/ [K+](i) ratio. Here, we report that the death of ouabain-treated epithelial cells from the Madin-Darby canine kidney (C7-MDCK) and endothelial cells from porcine aortae is suppressed by acidification of medium from pH 7.4 to 7.0, i.e. under conditions when pH(i) was decreased from similar to 7.2 to 6.9. The rescue of ouabain-treated C7-MDCK cells was also detected under selective intracellular acidification caused by inhibition of Na+/ H+ exchanger. In these cells, neither Na+, K+ pump activity nor [H-3]-ouabain binding was significantly affected by modest acidification. The death of ouabain-treated cells was independent of inhibition of RNA and protein synthesis with actinomycin D and cycloheximide. In contrast, both compounds sharply attenuated the protective action of acidified medium. Thus, our results show that very modest intracellular acidification is sufficient to inhibit the Na-i(+)/ K-i(+)-independent death signal triggered in epithelial and endothelial cells by CTS. They also suggest that the protective action of acidification is mediated by de novo expression of genes involved in inhibition of the cell death machinery.