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

Ouabain is a specific inhibitor of sodium, potassium-dependent adenosine tr iphosphatase (Na,K-ATPase), a P-type ion-transporting ATPase which is essen tial for the maintenance of adequate concentrations of intracellular Na+ an d K+ ions. The present study describes the establishment of a ouabain-resis tant mutant, TLouaR, from a human trophoblast cell line TL. Morphologically TL and TLouaR are indistinguishable, but, TLouaR is about 1000 times more resistant to the cytotoxic effect of ouabain and >2000 times to that of buf alin and yet ouabain can retard the growth of the TLouaR cells and in paral lel reduce its cloning efficiency in a time- and dose-dependent manner. Fur thermore, Na,K-ATPase activity from TLouaR cells is inhibitable by ouabain albeit with lower efficiency, [H-3]ouabain binding studies reveal that TLou aR cells have less (P < 0.05) ouabain binding sites (1.7 +/- 0.15 x 10(4)/c ell vs, 2.3+/-0.115 x 10(4)/cell in the control). However, affinities (diss ociation constants K-d) to ouabain for TL and TLouaR cells are not signific antly different. Lastly, Na,K-ATPase activity (1.375 +/- 0.25 <mu>mole ATP/ min . mg protein) of TLouaR cells is significantly higher (P < 0.05) than t hat of the TL cells (0.895 +/- 0.12 <mu>mole ATP/min . mg protein). These s tudies show that the interactions between ouabain and Na,K-ATPase can be me diated through different pathways resulting in diverse phenotypic character istics. In addition, ouabain resistance does not necessarily reflect the la ck of response to the digitalis drug. The exact mechanisms of ouabain resis tance observed in the present study remain to be determined but the TLouaR cells may be the best tool to uncover the many functional characteristics o f Na,K-ATPase.