Abstract
The uptake of [3H]formycin B by Ehrlich ascites tumor cells was examined in both normal Na+ buffer (physiological) and nominally Na+-free buffer (iso-osmotic replacement with Li+). These studies were conducted to further characterize the equilibrative nucleoside transporter subtypes of Ehrlich cells and to assess the contribution of Na+-dependent concentrative transport mechanisms to the cellular accumulation of nucleoside analogues by these cells. Formycin B is poorly metabolized by mammalian cells and, hence, can be used as a substrate to measure transport kinetics in energetically competent cells. Initial studies established that formycin B inhibited [3H]uridine uptake by the ei (equilibrative inhibitor-insensitive) and es (equilibrative inhibitor-sensitive) transporters of Ehrlich cells withKi values of 48 ± 28 and 277 ± 25 μM, respectively. Similarly, [3H]formycin B hadKm values of 111 ± 52 and 635 ± 147 μM for uptake by the ei and es transporters, respectively. When assays were conducted in the presence of Na+, plus 100 nM nitrobenzylthioinosine to prevent efflux via the es transporters, the intracellular concentration of [3H]formycin B exceeded the initial medium concentration by more than 3-fold, indicating the activity of a Na+-dependent transporter. Interestingly, the initial rate of uptake of [3H]formycin B was significantly higher in the Li+ buffer (es-mediated Vmax = 65 ± 10 pmol/μl · sec) than in the Na+buffer (Vmax = 8.4 ± 0.9 pmol/μl · sec); this may reflect trans-acceleration of [3H]formycin B uptake by elevated intracellular adenosine levels resulting from the low Na+ environment. This model was then used to assess the interaction of gemcitabine (2′,2′-difluorodeoxycytidine) with the equilibrative and concentrative nucleoside transporters. Gemcitabine, which has shown considerable potential for the treatment of solid tumors, was a relatively poor inhibitor of [3H]formycin B uptake via the equilibrative transporters (IC50 ∼ 400 μM). In contrast, gemcitabine was a potent inhibitor of the Na+-dependent nucleoside transporter of Ehrlich cells (IC50 = 17 ± 5 nM). These results suggest that the cellular expression/activity of Na+-dependent nucleoside transporters may be an important determinant in gemcitabine cytotoxicity and clinical efficacy.
Footnotes
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Send reprint requests to: Dr. James R. Hammond, Department of Pharmacology and Toxicology, Medical Sciences Building, The University of Western Ontario, London, Ontario, Canada N6A 5C1.
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↵1 This work was supported by a grant to J.R.H. from the Medical Research Council of Canada.
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↵2 es = e quilibrative, inhibitor s ensitive; ei = e quilibrative, inhibitor i nsensitive; cs = c oncentrative, inhibitor s ensitive;cif = c oncentrative, inhibitor i nsensitive, f ormycin B (purine) selective;cit = c oncentrative, inhibitor i nsensitive, t hymidine (pyrimidine) selective;cib = c oncentrative, inhibitor i nsensitive, b road substrate selectivity (nomenclature according to Belt et al., 1993).
- Abbreviations:
- NBMPR
- nitrobenzylthioinosine, nitrobenzylmercaptopurine riboside
- PBS
- phosphate-buffered saline
- ATP
- adenosine triphosphate
- Received August 15, 1997.
- Accepted April 27, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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