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 withK_i values of 48 ± 28 and 277 ± 25 μM, respectively. Similarly, [³H]formycin B hadK_m 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 V_max = 65 ± 10 pmol/μl · sec) than in the Na⁺buffer (V_max = 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.