Ouabain resistance of a human trophoblast cell line is not related to its reactivity to ouabain

Abstract

Ouabain is a specific inhibitor of sodium, potassium-dependent adenosine triphosphatase (Na,K-ATPase), a P-type ion-transporting ATPase which is essential for the maintenance of adequate concentrations of intracellular Na+ and K+ ions. The present study describes the establishment of a ouabain-resistant mutant, TL(ouaR), from a human trophoblast cell line TL. Morphologically TL and TL(ouaR) are indistinguishable, but, TL(ouaR) is about 1000 times more resistant to the cytotoxic effect of ouabain and > 2000 times to that of bufalin and yet ouabain can retard the growth of the TL(ouaR) cells and in parallel reduce its cloning efficiency in a time- and dose-dependent manner. Furthermore, Na,K-ATPase activity from TL(ouaR) cells is inhibitable by ouabain albeit with lower efficiency. [3H]ouabain binding studies reveal that TL(ouaR) cells have less (P < 0.05) ouabain binding sites (1.7 ± 0.15 x 104/cell vs. 2.3 ± 0.115 x 104/cell in the control). However, affinities (dissociation constants K(d)) to ouabain for TL and TL(ouaR) cells are not significantly different. Lastly, Na,K-ATPase activity (1.375 ± 0.25 μmole ATP/min·mg protein) of TL(ouaR) cells is significantly higher (P < 0.05) than that of the TL cells (0.895 ± 0.12 μmole ATP/min·mg protein). These studies show that the interactions between ouabain and Na,K-ATPase can be mediated through different pathways resulting in diverse phenotypic characteristics. In addition, ouabain resistance does not necessarily reflect the lack of response to the digitalis drug. The exact mechanisms of ouabain resistance observed in the present study remain to be determined but the TL(ouaR) cells may be the best tool to uncover the many functional characteristics of Na,K-ATPase.