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 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, 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 bufalin and yet ouabain can retard the growth of the TLouaR cells and in parallel reduce its cloning efficiency in a time- and dose-dependent manner. Furthermore, Na,K-ATPase activity from TLouaR cells is inhibitable by ouabain albeit with lower efficiency. [3H]ouabain binding studies reveal that TLouaR cells have less (P < 0.05) ouabain binding sites (1.7 +/- 0.15 x 10(4)/cell vs. 2.3 +/- 0.115 x 10(4)/cell in the control). However, affinities (dissociation constants Kd) to ouabain for TL and TLouaR cells are not significantly different. Lastly, Na,K-ATPase activity (1.375 +/- 0.25 micromole ATP/min mg protein) of TLouaR cells is significantly higher (P < 0.05) than that of the TL cells (0.895 +/- 0.12 micromole ATP/min x 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 TLouaR cells may be the best tool to uncover the many functional characteristics of Na,K-ATPase.
Ho
C K
CK
Department of Medical Research and Education, Veterans General Hospital, Taipei, Taiwan. cclin3@vghtpe.gov.tw
Chen
S M
SM
Wang
S Y
SY
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D002455:Cell Division; D002460:Cell Line; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D007928:Lethal Dose 50; D009154:Mutation; D010042:Ouabain / Q000494:pharmacology*; D010641:Phenotype; D011485:Protein Binding; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013997:Time Factors; D014327:Trophoblasts / Q000187:drug effects*
D004791:Enzyme Inhibitors; D010042:Ouabain; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000254:Sodium-Potassium-Exchanging ATPase