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 KD 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.


Supported by Grant CA-23100 from the National Cancer Institute, Grant CH-417 from the American Cancer Society, and Grant 100-R107 from Bristol-Myers, Co. This work was conducted in part by the Clayton Foundation for Research, California Division. P. A. A. is a Clayton Foundation Investigator.

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