Cardiotonic steroids differentially affect intracellular Na+ and [Na+]i/[K+]i-independent signaling in C7-MDCK cells

Recently, we reported that ouabain kills renal epithelial and vascular endothelial cells independently of elevation of the [Na+](i)/[K+](i) ratio. These observations raised the possibility of finding cardiotonic steroids (CTS) that inhibit the Na+, K+ pump without attenuating cell survival and vice versa. To test this hypothesis, we compared CTS action on Na+, K+ pump, [Na+](i) content, and survival of Madin-Darby canine kidney cells. At a concentration of 1 muM, ouabain and other tested cardenolides, as well as bufadienolides such as bufalin, cinobufagin, cinobufotalin, and telobufotoxin, led to similar to10-fold inhibition of the Na+, K+ pump, a 2-3-fold decrease in staining with dimethylthiazol-diphenyltetrazolium (MTT), and massive death indicated by detachment of similar to80% of cells and caspase-3 activation. In contrast, Na+, K+ pump inhibition and elevation of [Na+](i) seen in the presence of 3 muM marinobufagenin (MBG) and marinobufotoxin did not affect MTT staining and cell survival. Inhibition of the Na+, Rb+ pump in K+-free medium was not accompanied by a decline of MTT staining and cell detachment but increased sensitivity to CTS. In K+-free medium, half-maximal inhibition of Rb-86 influx was observed in the presence of 0.04 muM ouabain and 0.1 muM MBG, whereas half-maximal detachment and decline of MTT staining were detected at 0.03 and 0.004 muM of ouabain versus 10 and 3 muM of MBG, respectively. Both ouabain binding and ouabain-induced [Na+](i),[K+](i)-independent signaling were suppressed in the presence of MBG. Thus, our results show that CTS exhibit distinctly different potency in Na+, K+ pump inhibition and triggering of [Na+](i)/[K+](i)-independent signaling, including cell death.