Vol. 281, Issue 1, 347-353, 1997

Uptake and Release of [³H]Formycin B via Sodium-Dependent Nucleoside Transporters in Mouse Leukemic L1210/MA27.1 Cells¹

Stephanie L. Borgl and and Fiona E. Parkinson

Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, Manitoba, Canada

At least seven functionally distinct nucleoside transport processes exist; however, mouse leukemic L1210/MA27.1 cells possess only one subtype, a Na⁺-dependent transporter termed N1/cif. The capacity of this transporter subtype to release nucleosides from L1210/MA27.1 cells was investigated with the poorly metabolized inosine analog [³H]formycin B. Uptake of [³H]formycin B into these cells was inhibited by replacement of Na⁺ in the buffer with choline, or by blocking Na⁺/K⁺ ATPase with 2 mM ouabain, inhibiting glycolysis with 5 mM iodoacetic acid or inhibiting nucleoside transport with 1 mM phloridzin. Sodium stimulated uptake with an EC₅₀ value of 12 mM. To measure release of [³H]formycin B, cells were loaded with [³H]formycin B (10 µM) then washed and resuspended in buffer. Replacement of Na⁺ in the buffer with choline enhanced [³H]formycin B release by 20 to 47%, and significant stimulation of release was observed with Na⁺ concentrations of 30 mM or less. Resuspending loaded cells into Na⁺ buffer containing 2 mM ouabain or 10 µM monensin, a Na⁺ ionophore, significantly enhanced [³H]formycin B release during 20 min by 39% or 29%, respectively. Release of [³H]formycin B into choline buffer was inhibited 26.5% by 10 mM phloridzin and 39.6% by 10 mM propentofylline, compounds known to inhibit various transporters including Na⁺-dependent nucleoside transporters. Release was also inhibited significantly by 100 µM concentrations of dilazep, dipyridamole and nitrobenzylthioinosine, inhibitors with selectivity for Na⁺-independent nucleoside transporters. In the absence of Na⁺, the permeants adenosine and uridine enhanced [³H]formycin B release by up to 40.9% and 21.4%, respectively. These data indicate that in the absence of an inwardly directed Na⁺ gradient, Na⁺-dependent nucleoside transporters can function in the release of nucleosides.