Introduction

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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 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 [³H]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 transporter-mediated release of [³H]formycin B.

	Materials and Methods

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Materials. Mouse leukemic L1210/MA27.1 cells were provided by Dr. J.A. Belt. [³H]Formycin B was purchased from Moravek Biochemicals (Brea, CA). [³H]Adenosine, ³H₂O and [³H]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 × 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 ± 10 mOsm) then resuspended in Na⁺ buffer to 10⁶ 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 ± 10 mOsm with NaCl or choline chloride.

Measurements of [³H]formycin B uptake. [³H]Formycin B (10 µM; 6 µCi/ml) uptake into L1210/MA27.1 cells was measured by an oil-stop centrifugation method as described previously (Parkinson et al., 1993).

Measurements of [³H]formycin B release. Cells were washed and resuspended at 5 × 10⁶ cells/ml in Na⁺ buffer and loaded with 10 µM (1 µCi/ml) [³H]formycin B for 30 or 70 min at 37°C. To determine total cellular loading of [³H]formycin B, aliquots of cells (100 µl) were centrifuged (13,000 × g) through oil and associated radioactivity was determined. To assay cellular release of [³H]formycin B, 100-µl aliquots of cells were transferred to 1.5-ml microcentrifuge tubes, centrifuged (13,000 × 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 µl), and 400-µl aliquots were transferred to 1.5-ml microcentrifuge tubes containing 200 µl oil. After release intervals of 1 to 20 min, cells were centrifuged through oil and both supernatants (350 µl) 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%.

Measurements of [³H]adenosine release. The effect of iodoacetic acid on [³H]adenosine release was determined as described above, with cells loaded for 30 min (37°C) with [³H]adenosine (10 µM; 1 µCi/ml).

	Results

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Cellular accumulation of [³H]formycin B in L1210/MA27.1 cells. Cellular uptake of [³H]formycin B was greater with cells in Na⁺ buffer than with cells in choline buffer; the rates of uptake were 7.6 ± 0.3 pmol/10⁶ cells/min and 0.2 ± 0.4 pmol/10⁶ cells/min, respectively. For cells in Na⁺ buffer, uptake of [³H]formycin B was reduced by treatment of the cells with 2 mM ouabain, 5 mM iodoacetic acid or 1 mM phloridzin; the rates of uptake were 1.5 ± 0.2, 1.8 ± 0.4 and 0.6 ± 0.3 pmol/10⁶ cells/min, respectively (fig. 1). Uptake of [³H]formycin B was inhibited 23.6% by 100 µM NBMPR, 59.2% by 100 µM dilazep and 56.6% by 100 µM dipyridamole (data not shown). Sensitivity of [³H]formycin B uptake to Na⁺ was determined by measuring cellular accumulation in the presence of graded concentrations of NaCl. The EC₅₀ value obtained by nonlinear regression analysis was 12 mM Na⁺ (fig. 2).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Cellular accumulation of [3H]formycin B in L1210/MA27.1 cells. Cells were harvested, then washed and resuspended in Na+ (closed symbols) or choline (open squares) buffer. Cells were incubated with [3H]formycin B (10 µM) in buffer alone (squares) or in Na+ buffer containing 2 mM ouabain (closed triangles), 5 mM iodoacetic acid (closed circles) or 1 mM phloridzin (closed diamonds). Symbols represent means and bars represent S.E. for three separate experiments, each performed in quadruplicate.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Effect of extracellular Na+ concentration on [3H]formycin B uptake by L1210/MA27.1 cells. Cells were harvested, washed and then resuspended in buffer containing 0, 6, 12, 30, 59 or 118 mM NaCl. Choline chloride was added to maintain constant osmolarity. Uptake of [3H]formycin B during 180-sec intervals was measured. Symbols represent means and bars represent S.E. of three experiments performed in quadruplicate.

Release of [³H]formycin B from L1210/MA27.1 cells. Total [³H]formycin B loaded in 70 min was 99,000 ± 12,000 dpm/10⁶ cells (mean ± S.D.; n = 2). Release was stimulated by resuspending cells in Na⁺ or choline buffer at 22°C. During 10-min intervals, the percent of total loaded [³H]formycin B that was released into Na⁺ or choline buffer was 31 ± 4% (mean ± S.D.) or 53 ± 7%, respectively (fig. 3). The rate of release of [³H]formycin B at 22°C was 3.2 ± 0.3 pmol/5 × 10⁶ cells/min in choline buffer and 1.1 ± 0.2 pmol/5 × 10⁶ cells/min in Na⁺ buffer (fig. 3).

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Effect of extracellular Na+ on release of [3H]formycin B from L1210/MA27.1 cells. Cells were loaded with [3H]formycin B in Na+ buffer for 70 min (37°C). Cells were centrifuged briefly (5 sec, 13,000 × g) and extracellular [3H]formycin B was removed. Cells were resuspended in Na+ (filled circles) or choline (filled squares) buffer at 22°C. Release was terminated by centrifuging cells through oil. Symbols represent means and bars represent S.D. of two separate experiments, performed in quadruplicate.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Effect of Na+ concentration on release of [3H]formycin B from L1210/MA27.1 cells. Cells were incubated with [3H]formycin B for 30 min (37°C) in the presence of Na+. Cells were pelleted (5 sec; 13,000 × g) and extracellular [3H]formycin B was removed. Cells were resuspended in buffers containing 0, 30, 59 or 118 mM NaCl at 37°C and incubated for 10 (open bars) or 20 min (closed bars) before pelleting through oil (30 sec; 13,000 × g). Release of [3H]formycin B at 0 min was estimated with cells resuspended into buffers at 4°C and was 31.6 to 34.7 pmol. Values for 0 min were subtracted from values for 10- and 20-min release intervals. Bars represent S.E. of three separate experiments performed in quadruplicate. (*P < .05 ANOVA with Tukey's HSD post test comparing [3H]formycin B released in the presence of 0, 30 or 59 mM NaCl to [3H]formycin B released at 118 mM NaCl).

View larger version (11K): [in this window] [in a new window]   Fig. 5.   Effect of ouabain, monensin or iodoacetic acid on release of [3H]formycin B from L1210/MA27.1 cells. Cells were loaded with [3H]formycin B (10 µM) in Na+ buffer for 30 min (37°C). Cells were centrifuged briefly (5 seconds, 13,000 × g) and extracellular [3H]formycin B was removed. Cells were resuspended into Na+ buffer (37°C) in the absence (open bars) or presence (filled bars) of 2 mM ouabain (top panel), 10 µM monensin (center panel) or 5 mM iodoacetic acid (lower panel). Tritium content of supernatants was measured 10 or 20 min after resuspension. [3H]Formycin B content of supernatants at 0 min, determined by resuspending cells into Na+ buffer (4°C) in the absence or presence of ouabain, monensin or iodoacetic acid, was subtracted from 10- and 20-min values. Bars represent S.E. for three separate experiments performed in quadruplicate (*P < .05, paired t test comparing [3H]formycin B release in the presence and absence of inhibitor).

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Effect of iodoacetic acid on release of [3H]adenosine from L1210/MA27.1 cells. Cells were loaded with 10 µM [3H]adenosine in Na+ buffer for 30 min (37°C). Cells were centrifuged briefly (5 sec, 13,000 × g) and extracellular tritium was removed. Cells were resuspended into Na+ buffer (37°C) in the absence (open bars) or presence (filled bars) of 5 mM iodoacetic acid. Tritium content of supernatants was measured 10 or 20 min after resuspension. Tritium content of supernatants at 0 min was determined by resuspending cells into Na+ buffer (4°C) in the absence or presence of iodoacetic acid and was subtracted from values for 10- and 20-min release intervals. Bars represent S.E. for three separate experiments performed in quadruplicate. (*P < .05, **P < .01; paired t test comparing tritium release in the presence and absence of iodoacetic acid).

	Discussion

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The main finding of this study was that release of [³H]formycin B from L1210/MA27.1 cells was Na⁺ dependent; removal of extracellular Na⁺ or disruption of transmembrane Na⁺ gradients enhanced [³H]formycin B release.

As shown previously (Parkinson et al., 1993; Crawford et al., 1990a), uptake of nucleosides by mouse leukemic L1210/MA27.1 cells, was inhibited by removal of extracellular Na⁺. In the presence of physiological levels of Na⁺, the uptake of [³H]formycin B during a 5-min interval was 5-fold greater than in the absence of Na⁺. An EC₅₀ value of 12 mM Na⁺ was obtained, which agrees with the value (13 mM) for nucleoside transporter-mediated uptake of 6-mercaptopurine in rat intestinal brush-border membrane vesicles (Iseki et al., 1996). Phloridzin, an inhibitor of Na⁺-dependent transporters for glucose as well as those for nucleosides (Lee et al., 1988, 1990), inhibited [³H]formycin B uptake by 73% over 5 min. Disruption of transmembrane Na⁺ gradients by blocking Na⁺/K⁺ ATPase activity with ouabain or by depressing cellular ATP stores with the glycolytic inhibitor iodoacetic acid decreased [³H]formycin B uptake to 30 to 35% of control.

After loading of cells with [³H]formycin B, release was enhanced by removal of extracellular Na⁺, or by treating cells with phloridzin, ouabain or monensin, which indicated that nucleoside release from these cells is stimulated by conditions that perturb transmembrane Na⁺ gradients.

In contrast to the stimulatory effects of ouabain, monensin and Na⁺ replacement, the glycolytic inhibitor iodoacetic acid decreased [³H]formycin B release. By depressing intracellular ATP levels, iodoacetic acid can depress Na⁺/K⁺ ATPase activity and cause intracellular Na⁺ overload (Gemba et al., 1994); and it would thus be expected to have effects on nucleoside release similar to those of ouabain and monensin. We hypothesized that, by depressing ATP levels, iodoacetic acid elevated levels of intracellular adenosine which then competitively inhibited release of [³H]formycin B. Consistent with this hypothesis, we found that iodoacetic acid stimulated tritium release in cells loaded with [³H]adenosine. The difference in release of these two compounds indicates that [³H]adenosine is the better permeant for outward transport. Previously, it had been shown that Na⁺-dependent influx of 1 µM adenosine (190 pmol/10⁹ cells/sec) was approximately 8-fold faster than that of 1 µM formycin B (24 pmol/10⁹ cells/sec) in L1210 cells (Crawford et al., 1990b) and that adenosine has greater affinity than formycin B for N1/cif transporters (Vijayalakshmi and Belt 1988).

An interesting finding of these studies was that treatment of cells with phloridzin, ouabain or Na⁺-replacement buffer was more effective in inhibiting [³H]formycin B uptake than in stimulating [³H]formycin B release. At least three factors may contribute to this difference. First, each of these treatments may elevate intracellular adenosine levels. In this case, total nucleoside release may be underestimated by measuring [³H]formycin B release, because simultaneous release of nonradioactive adenosine may competitively inhibit [³H]formycin B release. Second, uptake studies were performed with cells pretreated with the desired buffers and drugs; however, because pretreatment was not possible for release studies, release was measured from the beginning of exposure of cells to the various treatment conditions. Because the drugs were not at equilibration with their respective target sites before initiation of release, this could lead to underestimation of the effects of the cell treatments on [³H]formycin B release. Third, the finite intracellular volume of the cells meant that intracellular [³H]formycin B concentrations were not constant for the duration of the release intervals. Each of these three factors would have the effect of lowering [³H]formycin B release.

Differences were also observed in the Na⁺ concentration dependence of [³H]formycin B uptake and release; for example, uptake was unaffected but release was stimulated by reducing the buffer Na⁺ concentration from physiological to 30 mM. This may indicate that intracellular levels of Na⁺ are higher in cells used for release assays than in cells used for uptake assays. It is possible that intracellular Na⁺ levels are elevated before initiation of release intervals, because cells are loaded with [³H]formycin B in the presence of Na⁺ buffer.

Release of [³H]formycin B was depressed by millimolar concentrations of low-affinity inhibitors of Na⁺-dependent nucleoside transporters, such as propentofylline (Parkinson et al., 1993) and phloridzin (Lee et al., 1988, 1990). Release was also decreased by 10 to 100 µM concentrations of NBMPR, dipyridamole and dilazep, inhibitors that at nanomolar concentrations are selective for Na⁺-independent nucleoside transporters (Cass, 1995). Several studies have measured adenosine release in the presence or absence of NBMPR or dipyridamole at concentrations of 10 to 100 µM (Hoehn and White, 1990; Craig and White, 1993; Green, 1980; Cunha et al., 1996). Inhibition of release has been interpreted as evidence of release mediated by equilibrative transporters. However, the present study indicates that NBMPR, dipyridamole and dilazep can inhibit nucleoside uptake and release mediated by Na⁺-dependent transporters. Thus, high (>10 µM) concentrations of these compounds should be used with caution in investigations of cellular release mechanisms for nucleosides.

Stimulation of release by adenosine and uridine may indicate transacceleration in the absence of a Na⁺ gradient. This phenomenon, commonly observed with Na⁺-independent nucleoside transporters (Jarvis, 1986), can occur when transporter permeants are simultaneously present on both sides of the membrane. In the presence of a Na⁺-gradient, Na⁺-dependent transporters function as symporters and translocate nucleosides in an inward direction. As long as the Na⁺ gradient is maintained, the intracellular accumulation of permeants does not appear to affect permeant uptake. Our data suggest, however, that disruption of transmembrane Na⁺ gradients may uncouple nucleoside transport from Na⁺ translocation, and in this situation transport of nucleosides in one direction may accelerate the transfer in the opposite direction.

Carrier-mediated release of neurotransmitters, including glutamate, -aminobutyric acid and dopamine, has been demonstrated by elevating intracellular Na⁺ levels, replacing extracellular Na⁺, blocking Na⁺/K⁺ ATPase activity or inhibiting glycolysis (Gemba et al., 1994; Eshleman et al., 1994; Levi and Raiteri, 1993; Belhage et al., 1993). Furthermore, it has been suggested that carrier-mediated release of glutamate is a significant source of excitotoxic extracellular glutamate in cerebral ischemia (Szatkowski and Attwell, 1994). Adenosine released via reversal of Na⁺-dependent nucleoside transporters may contribute to the micromolar levels of extracellular adenosine that arise during cerebral ischemia. Molecular evidence indicates that mRNA for N1/cif and N2/cit transporters is widely distributed in brain (Anderson et al., 1996). Other sources that may contribute to elevated extracellular adenosine levels include release via Na⁺-independent transporters and release of ATP followed by enzymatic dephosphorylation to adenosine.

In summary, we have demonstrated that by disrupting transmembrane Na⁺ gradients, reversal of Na⁺-dependent nucleoside transporters can mediate cellular release of nucleosides. The evidence that this release is transporter-mediated includes inhibition by transport inhibitors and stimulation by transporter permeants. Adenosine, a nucleoside with diverse receptor-mediated effects, may be released from cells by this process during conditions, such as ischemia, that depress cellular transmembrane Na⁺ gradients by compromising intracellular ATP levels and/or Na⁺/K⁺ ATPase function.