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

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 [H]formycin B. Uptake of [H]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 [H]formycin B, cells were loaded with [H]formycin B (10 mM) then washed and resuspended in buffer. Replacement of Na in the buffer with choline enhanced [H]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 mM monensin, a Na ionophore, significantly enhanced [H]formycin B release during 20 min by 39% or 29%, respectively. Release of [H]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 mM 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 [H]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. Nucleoside transport processes are membrane-bound carrier proteins that mediate the transfer of nucleosides across plasma membranes. Seven transporters have been characterized according to function (Cass, 1995) and are divided into two broad classes: Na-independent and Na-dependent processes. Na-independent transporters are facilitated diffusion processes that catalyze cellular influx or efflux of nucleosides with the direction of movement determined by the nucleoside concentration gradient. Two equilibrative transporters are distinguished by their sensitivity to the transport inhibitor NBMPR and are termed equilibrative sensitive (es) and equilibrative insensitive (ei), respectively (Vijayalakshmi and Belt, 1988). Na-dependent transporters couple the influx of Na to the influx of nucleosides; thus, in the presence of a transmembrane Na-gradient nucleosides can be concentrated within cells to levels in excess of those in the extracellular environment. Five Na-dependent nucleoside transporters have been described and are termed N1 to N5. N1, also called cif, accepts purines and uridine as permeants, whereas N2, also called cit, and N4 are pyrimidine selective. N3 and N5, also called cib and cs, respectively, have broad permeant selectivity and accept both purines and pyrimidines. N5 (cs) is unique among the currently identified Na-dependent transporters for its sensitivity to inhibition by low nanomolar concentrations of NBMPR. Dipyridamole and dilazep inhibit both es and ei but are poor inhibitors of Na-dependent transporters (Cass, 1995). Nucleoside transport processes are an important component of nucleoside salvage pathways and provide cells with nucleosides that are required for cellular metabolism. In addition, adenosine is an endogenous nucleoside that has autocrine and paracrine regulatory effects. In brain, adenosine is an inhibitory neuromodulator, and extracellular adenosine levels are regulated by nucleoside transport processes. Recent evidence indicates that glutamate transporters, which are dependent on Na and normally function in cellular uptake, can mediate glutamate release after depolarization, ATP depletion or glycolytic inhibition (Madl and Burgesser, 1993; Gemba et al., 1994). It has been proposed that Received for publication August 26, 1996. 1 This work was supported by the Medical Research Council of Canada (MRCC). F.E.P. is a Scholar of the MRCC. ABBREVIATIONS: NBMPR, nitrobenzylthioinosine or nitrobenzylmercaptopurine riboside. 0022-3565/97/2811-0347$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 281, No. 1 Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 281:347–353, 1997 347 at A PE T Jornals on O cber 5, 2017 jpet.asjournals.org D ow nladed from this is an important source of extracellular glutamate during conditions of abnormal metabolism, such as stroke (Szatkowski and Attwell, 1994). Because adenosine levels also increase during stroke and cellular release of adenosine can be resistant to inhibitors of es and ei transporters (Geiger and Fyda, 1991), we investigated whether Na-dependent nucleoside transporters can mediate nucleoside release during conditions that perturb transmembrane Na gradients. Murine leukemia L1210 cells possess both Na-independent (es and ei) and Na-dependent (N1/cif) nucleoside transporter activities (Crawford et al., 1990b). Mutation strategies led to the isolation of L1210/MA27.1 cells which retain only an N1/cif nucleoside transporter (Crawford et al., 1990a); thus, these cells provide a model system to examine the function of Na-dependent nucleoside transporters. We investigated cellular release of [H]formycin B, a poorly metabolized inosine analog (Plagemann et al., 1990; Dagnino and Paterson, 1990; Wu et al., 1993) that is a permeant of N1/cif transporters present in L1210/MA27.1 cells (Crawford et al., 1990a), and found evidence for Na-dependent transportermediated release of [H]formycin B. Materials and Methods Materials. Mouse leukemic L1210/MA27.1 cells were provided by Dr. J.A. Belt. [H]Formycin B was purchased from Moravek Biochemicals (Brea, CA). [H]Adenosine, H2O and [ H]polyethylene glycol were from DuPont NEN (Boston, MA). NBMPR was obtained from Research Biochemicals International (Natick, MA). RPMI 1640 and heat-inactivated horse serum were purchased from Gibco BRL (Burlington, Ontario). Dilazep was provided by F. Hoffmann-LaRoche Ltd (Basel, Switzerland). All other reagents were obtained from Sigma Chemical Co. (St. Louis, MO). Cell culture. Mouse leukemic L1210/MA27.1 cells were maintained in logarithmic phase growth in RPMI 1640 culture medium with 10% heat-inactivated horse serum. Cells were harvested by centrifugation at 100 3 g for 10 min, washed twice with Na buffer (in mM: NaCl, 118; KCl, 4.9; MgCl2, 1.2; KH2PO4, 1.4; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 25; glucose, 11; CaCl2, 1; pH 7.4, 300 6 10 mOsm) then resuspended in Na buffer to 10 cells/ml. For some experiments, cells were washed and resuspended in buffer in which NaCl was replaced with equimolar choline chloride (choline buffer). For experiments with iodoacetic acid, glucose was omitted from the buffer. Osmolarity of buffers was adjusted, as necessary, to 300 6 10 mOsm with NaCl or choline chloride. Measurements of [H]formycin B uptake. [H]Formycin B (10 mM; 6 mCi/ml) uptake into L1210/MA27.1 cells was measured by an oil-stop centrifugation method as described previously (Parkinson et al., 1993). The effect of ouabain, an inhibitor of Na/K ATPase, iodoacetic acid, an inhibitor of glycolysis, or phloridzin, an inhibitor of Nadependent nucleoside transport (Lee et al., 1990), on [H]formycin B uptake was assessed. Cells were preincubated with 2 mM ouabain for 40 min at 37°C (Dagnino et al., 1991), 5 mM iodoacetic acid for 20 min at 37°C (Plagemann and Aran, 1990) or 1 mM phloridzin for 15 min at 22°C (Huang et al., 1993) and [H]formycin B uptake (22°C) was determined. The effect of nucleoside transport inhibitors on [H]formycin B uptake was determined with cells preincubated for 15 min (22°C) with 100 mM concentrations of NBMPR, dilazep or dipyridamole. The effect of graded Na concentrations on [H]formycin B uptake was determined by preparing and incubating (15 min, 22°C) cells in buffers containing 0, 6, 12, 30, 59 or 118 mM NaCl. Aliquots of cells were added to reaction mixtures containing [H]formycin B in identical Na concentrations. After uptake intervals of 180 sec, reactions were terminated and cell-associated radioactivity was determined. Measurements of [H]formycin B release. Cells were washed and resuspended at 5 3 10 cells/ml in Na buffer and loaded with 10 mM (1 mCi/ml) [H]formycin B for 30 or 70 min at 37°C. To determine total cellular loading of [H]formycin B, aliquots of cells (100 ml) were centrifuged (13,000 3 g) through oil and associated radioactivity was determined. To assay cellular release of [H]formycin B, 100-ml aliquots of cells were transferred to 1.5-ml microcentrifuge tubes, centrifuged (13,000 3 g) for 5 sec and loading buffer was aspirated. Cell pellets were cooled on ice and then resuspended in either Na or choline buffer (22°C; 500 ml), and 400-ml aliquots were transferred to 1.5-ml microcentrifuge tubes containing 200 ml oil. After release intervals of 1 to 20 min, cells were centrifuged through oil and both supernatants (350 ml) and cell pellets were analyzed for radioactivity. Cells resuspended into buffer at 4°C were used to estimate release at 0 min. Cell viability after resuspension was determined by trypan blue exclusion assays and was routinely greater than 95%. The effect of extracellular Na concentrations on [H]formycin B release was determined by resuspending [H]formycin B-loaded cells in 4°C or 37°C buffer containing 0, 30, 59 or 118 mM NaCl. Values of release at 0 min were subtracted from 10and 20-min release values for each buffer. To determine the effects of ouabain, iodoacetic acid or the Naionophore monensin on [H]formycin B release, cells loaded for 30 min with [H]formycin B were resuspended in Na buffer (4°C or 37°C) alone or in Na buffer containing 2 mM ouabain, 10 mM monensin or 5 mM iodoacetic acid. Release of [H]formycin B during time intervals of 0, 10 or 20 min was measured as described above. To test whether these treatments affected cell viability, trypan blue dye exclusion or intracellular water volume was measured. To determine intracellular volume, cells were incubated in Na buffer for 30 min at 37°C, centrifuged and resuspended in buffer as described above. After 20 min at 37°C, H2O (0.7 mCi/ml) or [ H]polyethylene glycol (0.7 mCi/ml) was added and cells were incubated for a further 3 min. Cells were then centrifuged through oil and cell pellets were assayed for tritium content. The effects of inhibitors or permeants of nucleoside transport processes on release of [H]formycin B were evaluated. Cells were loaded with [H]formycin B in Na buffer for 30 min at 37°C. Cell aliquots (100 ml) were centrifuged (13,000 3 g) for 5 sec, supernatants were removed and pellets were resuspended in 500 ml choline buffer in the absence or presence of the nucleoside transport inhibitor phloridzin, dilazep, dipyridamole, NBMPR or propentofylline, or in the absence or presence of the N1/cif transporter permeant adenosine or uridine. Cells were incubated for 10 or 20 min at 37°C and then centrifuged through oil. Measurements of [H]adenosine release. The effect of iodoacetic acid on [H]adenosine release was determined as described above, with cells loaded for 30 min (37°C) with [H]adenosine (10 mM; 1