Role of the Na⁺,K⁺-Adenosine Triphosphatase in the Accumulation of cis-Diamminedichloroplatinum(II) in Human Ovarian Carcinoma Cells¹ Free

We examined the importance of the Na⁺,K⁺-ATPase in cisplatin (DDP) accumulation in 2008 human ovarian carcinoma cells and describe changes in the Na⁺,K⁺-ATPase in DDP-resistant cells with DDP accumulation defects. Approximately 50% of DDP accumulation was inhibitable by ouabain. DDP accumulation into 2008 cells could be maximally inhibited when cells were preincubated with ouabain for 1 h prior to DDP exposure. The half-maximal inhibition was obtained with 0.13 µm ouabain. Similar inhibition of DDP accumulation was obtained when the Na⁺,K⁺-ATPase was blocked by ATP depletion or by incubating cells in K⁺-free medium. This same percentage of DDP accumulation was Na⁺ dependent and varied directly with Na⁺ concentration. These effects on DDP accumulation could be detected as early as 1 min after the imposition of 0-trans conditions, strongly suggesting that the inhibition was due to modulation of a drug influx step. The Na⁺,K⁺-ATPase in 2008/DDP cells had a similar K_D for ouabain binding and 36% less Na⁺,K⁺-ATPase molecules/mg of protein than 2008 cells. 2008/DDP cells were 2.3 ± 0.2 (SE, n = 3) fold cross-resistant to ouabain in a continuous exposure clonogenic assay. Despite these changes in the Na⁺,K⁺-ATPase, the net basal Na⁺,K⁺-ATPase activity was the same in sensitive and DDP-resistant cells as determined by ouabain-inhibitable ⁸⁶Rb⁺ influx. The basal Na⁺ levels were also similar in the sensitive and resistant cells. These data suggest that DDP accumulation is partially Na⁺ dependent and that, therefore, the Na⁺,K⁺-ATPase which maintains the Na⁺ gradient may play an important role in determining how much DDP enters cells. Whether there is a causal link between the changes in the Na⁺,K⁺-ATPase in DDP-resistant cells and their DDP accumulation defect is not yet known.