Characterization of a Nucleoside/Proton Symporter in Procyclic Trypanosoma brucei brucei*

Harry P. de Koning, Christopher J. WatsonDagger , and Simon M. Jarvis§

From the Research School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, United Kingdom

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Adenosine transport at 22 °C in procyclic forms of Trypanosoma brucei brucei was investigated using an oil-inhibitor stop procedure for determining initial rates of adenosine uptake in suspended cells. Adenosine influx was mediated by a single high affinity transporter (Km 0.26 ± 0.02 µM, Vmax 0.63 ± 0.18 pmol/107 cells s-1). Purine nucleosides, with the exception of tubercidin (7-deazaadenosine), and dipyridamole inhibited adenosine influx (Ki 0.18-5.2 µM). Purine nucleobases and pyrimidine nucleosides and nucleobases had no effect on adenosine transport. This specificity of the transporter appears to be similar to the previously described P1 adenosine transporter in bloodstream forms of trypanosomes. Uptake of adenosine was Na+-independent, but ionophores reducing the membrane potential and/or the transmembrane proton gradient (monitored with the fluorescent probes bis-(1,3-diethylthiobarbituric acid)-trimethine oxonol and 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester, respectively) inhibited adenosine transport. Similarly, an increase in extracellular pH from 7.3 to 8.0 reduced adenosine influx by 30%. A linear correlation was demonstrated between the rate of adenosine transport and the protonmotive force. Adenosine uptake was accompanied by a proton influx in base-loaded cells and was also shown to be electrogenic. These combined results indicate that transport of adenosine in T. brucei brucei procyclics is protonmotive force-driven and strongly suggest that the adenosine transporter functions as an H+ symporter.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Parasitic protozoa of the Trypanosoma brucei subgroup are the causative agents of African sleeping sickness in man and the related livestock disease, nagana. Parasitic protozoa, including T. brucei brucei, are unable to synthesize their own purines (1), relying on the salvage of preformed purines from the host environment to satisfy their nucleotide requirements. Salvage consists of intracellular and extracellular metabolic events (2, 3) plus the permeation of the substrate across the plasma membrane of the parasite. Whereas extensive studies of purine metabolism in the trypanosomatidae have been carried out (for review see Ref. 1), the mode by which purines are taken up from the environment by T. brucei has received limited attention. The first report on the uptake of nucleosides by T. brucei appeared in 1980 (4). Adenosine uptake was suggested to occur in a manner consistent with two mechanisms, one with high affinity and the other with low affinity for adenosine. However, this study used relatively long incubation times (3 min at 37 °C) which did not correspond to initial rate conditions. More recently, rapid initial adenosine transport fluxes were determined over a period of 0-5 s in bloodstream forms of T. brucei at 25 °C (5). Two high affinity adenosine transporters (P1 and P2) were identified with apparent Km values for adenosine influx of 0.15 and 0.59 µM, respectively (5). The P1 system was selectively inhibited by inosine, whereas the P2 transporter was specifically blocked by adenine. Interestingly, one of these carriers (P2) appeared to transport melamino-phenylarsenicals, of which melarsoprol is a front-line drug in the treatment of late stage sleeping sickness (6, 7). Moreover, melarsen-resistant trypanosomes lacked a functional P2 adenosine transport system (5). Nevertheless, the mechanism of either P1 or P2 adenosine transport is unknown, and the substrate specificity of the two carriers was not fully characterized.

During their life cycle, T. brucei live alternatively in the bloodstream of mammalian hosts and in the digestive tracts of tsetse flies. In contrast to the bloodstream forms of T. brucei, nothing is known regarding how nucleosides are transported by procyclic cultured forms, homologous to the insect midgut stage. Given the importance of purine transport to the parasite and the possible involvement of this process in drug uptake and resistance, there is a need for a thorough understanding of nucleoside transport in all forms of this organism. In the present study, a rapid inhibitor oil-stop technique was utilized to investigate adenosine transport in cultured procyclic T. brucei brucei. We show that procyclic forms of T. brucei brucei possess a single adenosine transporter that apparently accepts only other purine nucleosides and appears to be similar to the P1 adenosine transporter in bloodstream forms of T. brucei brucei. Moreover, we demonstrate that the adenosine transporter in procyclic forms is protonmotive force-driven.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Trypanosome Culture-- Procyclic forms of T. brucei brucei strain 427 were grown as described by Brun and Schönenberger (8) in SDM-79 medium. Cells at mid-logarithmic stage of growth were harvested and washed twice in assay buffer (33 mM Hepes, 98 mM NaCl, 4.6 mM KCl, 0.3 mM CaCl2, 0.07 mM MgSO4, 5.8 mM NaH2PO4, 0.3 mM MgCl2, 23 mM NaHCO3 and 14 mM glucose, pH 7.3). Cells were resuspended in buffer to approximately 108 cells/ml.

Nucleoside Transport Measurements-- All measurements and dilutions were performed in assay buffer. Transport was measured by an adaptation of a rapid oil-stop method (9). An oil layer was utilized for the separation of cells from incubation medium after transport was stopped by addition of a high concentration of ice-cold unlabeled permeant. The oil used had a density of 1.018 g/ml, composed by mixing 7 volumes of dibutylphthalate (1.043 g/ml) and 1 volume of mineral oil (0.84 g/ml).

Assay mixtures were prepared in 1.5-ml microcentrifuge tubes. Added first was 200 µl of oil upon which was layered 100 µl of transport medium containing 3H-nucleoside (10 µCi/ml). Transport was initiated by addition of 100 µl of procyclic T. brucei brucei cells (approx 107). All transport assays were performed at 22 °C and in triplicate. Intervals of transport were terminated by the addition of 1 ml of ice-cold stop solution (assay buffer containing 4 mM unlabeled permeant), followed by immediate centrifugation for 30 s at 12,000 × g. To determine the amount of accumulated radioactivity associated with the cell pellet, oil and aqueous layers were removed by suction, and the inside of the tube was wiped with absorbent paper, and the pellet was dissolved in 0.5 M NaOH. Samples were then left overnight. Subsequently, 1 ml of scintillation fluid (Optiphase Hisafe III) was added, and samples were counted in a Beckman LS 6000 TA scintillation counter. Blank values (radioactivity that became associated with cells during uptake intervals of 0 s) were obtained by processing cell samples exposed simultaneously to 3H-permeant and 4 mM unlabeled permeant. Where applicable, kinetic constants of influx (apparent Km and Vmax) and inhibition constants (IC50 values and apparent Ki) were determined by nonlinear regression analysis of data using appropriate equations and the computer programs Enzfitter (Elsevier Biosoft) and Inplot (Graphpad Software).

In inhibition studies, test compounds and [3H]adenosine or [3H]inosine were added to cells simultaneously, except metabolic inhibitors and ionophores that were preincubated with cells for 1-10 min before addition of [3H]adenosine. Stock solutions of metabolic inhibitors and ionophores were made in ethanol, and control studies performed in parallel confirmed that adenosine transport rates were not affected by the final concentration of ethanol (<2%). Transport rates were expressed per 107 cells, and the cell number in suspensions of T. brucei brucei was enumerated using a hemocytometer observed under phase contrast microscopy. The intracellular volume of procyclic forms of T. brucei brucei was determined from the distribution ratio at equilibrium of [3H]H2O with either [14C]sucrose or [14C]mannitol as the extracellular marker. Cells were mixed with the two isotopes for 0.5 min and then separated from the extracellular medium by directly centrifuging through the oil layer without the addition of cold stop as described above. Uptake rates as well as Km and Ki values are given ±S.E. unless otherwise indicated.

Metabolism-- To explore the metabolic fate of transported substrate, the inert oil layer was underlayered with 100 µl of 20% (w/v) perchloric acid. The assay was performed as described for the transport experiments, and cells were spun through the oil layer into the perchloric acid. Upper oil and aqueous layers were removed, and lysate was neutralized with 5 M KOH. Precipitate was removed by centrifugation (2 min, 12,000 × g), and aliquots of the supernatant were subjected to thin layer chromatographic analysis using 0.2-mm plastic sheets coated with silica gel (Merck) in a butan-1-ol, ethyl acetate, methanol, and ammonia (7:4:3:4) solvent system (Rf values of 0, 0.53, 0.63, 0.46, and 0.36 for ATP, adenosine, adenine, hypoxanthine, and inosine, respectively). Bands were located under UV light and scraped into vials for scintillation counting. Greater than 85% of the radioactivity loaded onto the TLC plates was recovered.

Fluorescent Measurements-- To load procyclic T. brucei brucei with the pH-sensitive fluorescent dye BCECF,1 cells were washed twice in assay buffer, resuspended in the same buffer containing 5 µM BCECF/AM, and incubated at 25 °C for 1 h at a density of approximately 108 cells/ml. Cells were pelleted and washed twice with assay buffer to remove extracellular dye and resuspended at a final cell density of 108 cells/ml until used. A 3-ml aliquot of BCECF-loaded procyclic cells was transferred to a 1-cm square quartz cuvette and maintained at 25 °C. Fluorescence measurements were carried out in a Perkin-Elmer LS 50B fluorimeter. Excitation (440 and 490 nm) and emission (530 nm) were set at 10-nm bandwidths. The 490 and 440 nm fluorescence intensity signals and the 490/440 ratio signals were continuously monitored. Calibration of intracellular pH (pHi) versus fluorescence was carried out using 20 µM nigericin in a K+-rich Ringers solution (20 mM KCl, 10 mM NaCl, 2 mM MgCl2, 100 mM potassium gluconate, 10 mM glucose, 5 mM Mes, and 5 mM Mops) at several different pH levels from 6.2-8.0 as described (10). Intracellular fluorescence was predominantly localized in the parasite cytosol, as visualized by confocal microscopy. Membrane potential (Vm) was measured and calibrated using the fluorescent anionic probe bis-oxonol as described (10), with excitation at 540 nm and emission recorded at 580 nm. Briefly, 100 µl of procyclic cells (107) or assay buffer were added to 2.9 ml of 0.1 µM bis-oxonol in assay buffer in a cuvette in the fluorimeter. Fluorescence was recorded, and the Vm was derived using the difference in intensity between the two traces, and a calibration curve was obtained by the method of Vieira et al. (11), in the presence of gramicidin. For every trace where permeants or ionophores were added to cells, a similar trace was obtained in the absence of cells and subtracted as background. In addition, control traces were recorded adding assay buffer or ethanol (solvent for CCCP) instead of inhibitor. The protonmotive force (PMF) was calculated from Equation 1 (12).
<UP>PMF</UP>=V<SUB>m</SUB>−(2.3RT/F)(<UP>pH</UP><SUB>i</SUB>−<UP>pH</UP><SUB>o</SUB>) (Eq. 1)
in which pHo is the extracellular pH.

ATP Measurements-- Intracellular measurements of ATP were determined using a bioluminescent assay, as described previously (10).

Materials-- Nucleosides, nucleobases, and ionophores were purchased from Sigma. [2,8,5'-3H]Adenosine (2.3 TBq/mmol) was obtained from NEN Life Science Products. [3H]Inosine (1.4 TBq/mmol) was purchased from Moravek Biochemicals. [3H]H2O (925 MBq/g), [14C]sucrose (23.3 GBq/mmol), D-[1-14C]mannitol (2.11 GBq/mmol), and [3H]thymidine (3 TBq/mmol) were obtained from Amersham Pharmacia Biotech. BCECF and bis-oxonol were purchased from Calbiochem and Molecular Probes, respectively. Cell culture reagents were obtained from Life Technologies, Inc. All other reagents were analytical grade.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Adenosine Transport Measurements-- Studies of nucleoside transport in suspended mammalian cells have utilized oil-stop methods that are capable of resolving time courses of uptake during the first few seconds of exposure of cells to permeant (13). Two principal methods have been used. The first technique relies on the termination of uptake by simply centrifuging the cells through an oil layer (14, 15). This process, when estimated in mammalian cells, takes approximately 2 s for the complete separation of cells from the extracellular radioactive permeant (14, 15). This technique has been adopted by a variety of laboratories to determine initial transport rates of various molecules in parasites (5, 16, 17). The second method uses the addition of excess buffer containing a transport inhibitor to block transport followed by centrifugation of cells under the oil layer (9, 15). Time courses of adenosine influx by T. brucei brucei procyclic cells using the two different stopping methods are shown in Fig. 1A. Intervals of adenosine uptake were ended by (a) pelleting the cells under the oil layer or (b) addition of ice-cold 4 mM adenosine, followed immediately by centrifugation of cells through the oil layer. Adenosine uptake was rapid and linear for at least 20 s, and the slopes of uptake by both methods were identical. The lack of curvature in these plots would suggest initial transport rates are being measured. However, the intercept on the ordinate for the two time courses was markedly different. In the presence of 4 mM adenosine, the time course intercepted with the estimate of extracellular space. Without the addition of unlabeled adenosine, [3H]adenosine uptake appeared to continue during the centrifugation step and was equivalent to uptake over a 4-s period as evident from the intercept with the extracellular fluid of -4 s. Thus procyclic forms of T. brucei brucei take considerably longer to pellet through the oil than the usual 2-s lag estimated for mammalian cells. In further experiments, addition of ice-cold excess permeant was used to terminate transport.


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Fig. 1.   Adenosine uptake in T. brucei brucei procyclics. A, time course of adenosine influx. Transport at 22 °C was initiated by the addition of 100 µl of procyclic cells (107 cells) to 100 µl of transport medium containing 2 µM [3H]adenosine. After the appropriate time interval, transport was terminated by either centrifugation alone (square ) or by addition of 1 ml of assay buffer containing 4 mM adenosine followed by immediate centrifugation (black-square). B, concentration dependence of adenosine influx. Procyclic cells were incubated with graded concentrations (0.1-5 µM) of [3H]adenosine for varying time intervals between 2 and 10 s. Initial velocities of adenosine influx were calculated from linear regression analysis of the time course data for each adenosine concentration. The kinetic constants were determined by nonlinear regression analysis using the Michaelis-Menten equation and gave a Km value of 0.19 ± 0.04 µM and a Vmax of 0.45 ± 0.02 pmol (107 cells)-1 s-1 (± S.E.).

Metabolism of Permeant-- Metabolism of 1 µM [3H]adenosine was monitored by TLC following an incubation period of 0, 5, and 10 s. After 5 s 7% of the recovered radioactivity was associated with adenosine with the majority of label (88%) recovered in the nucleotide fraction. After 10 s the label in the latter fraction had increased slightly to 92%. Based on the measured intracellular volume of T. brucei brucei procyclics (0.48 ± 0.02 µl per 107 cells; S.D., n = 3), we calculate that the small amount of unmetabolized adenosine present did not exceed the concentration of that found extracellularly. The practical consequence of this rapid intracellular phosphorylation is that the transmembrane gradient of adenosine is maintained over the period of 10s of seconds and thus initial rates of transport in T. brucei brucei procyclics can be determined with an interval of 10 s (see Fig. 1A and Ref. 18).

Kinetic Parameters of Adenosine Transport-- Initial rates of adenosine transport were measured over the concentration range 0.05-5 µM for [3H]adenosine. Fig. 1B shows that adenosine influx was saturable and conformed to simple Michaelis-Menten kinetics (Km 0.19 ± 0.04 µM; Vmax 0.45 ± 0.02 pmol/107 cells s-1). The mean values from three separate experiments were 0.26 ± 0.02 µM and 0.63 ± 0.18 pmol/107 cells s-1 for Km and Vmax, respectively.

Substrate Specificity of the Adenosine Transporter-- The substrate specificity of adenosine transport in T. brucei brucei procyclic forms was studied by investigating the effect of a variety of compounds on the initial rates of adenosine influx. The inhibition profiles obtained with unlabeled adenosine, guanosine, and thymidine are shown in Fig. 2A. Adenosine and guanosine totally inhibited [3H]adenosine influx in a monophasic manner with Hill slopes of 1.2 ± 0.4 and 0.94 ± 0.1, respectively. These findings are consistent with the suggestion that both nucleosides are permeants for the same carrier. The apparent Ki value derived from this data for self-inhibition of adenosine influx was 0.25 ± 0.05 µM (n = 3), a value similar to the apparent Km estimate. In contrast, thymidine had no significant effect on adenosine influx by T. brucei brucei procyclics at concentrations up to 100 µM. In a parallel experiment, the flux of 1 µM [3H]thymidine (0.014 pmol/107 cells s-1) was determined to be 50-fold less than the corresponding rate of adenosine transport at the same concentration.


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Fig. 2.   Inhibition of adenosine and inosine transport in T. brucei brucei procyclics. A, uptake of 1 µM [3H]adenosine at 5 s was determined in the presence of thymidine (black-down-triangle ), adenosine (black-square), and guanosine (open circle ). The results are the average of three separate experiments (± S.E.) and are presented as a percentage of the control influx rate (0.7 pmol (107 cells)-1 s-1) (see Table I for Ki values). B, uptake of 50 nM [3H]inosine at 10 s was determined in the presence of adenosine (black-square), guanosine (open circle ), tubercidin (triangle ), and uracil (black-triangle). The results are the average of triplicate determinations (± S.E.) and are representative of three similar experiments. Ki values for the adenosine and guanosine traces shown were 0.20 ± 0.003 and 0.28 ± 0.02 µM, respectively.

The studies shown in Fig. 2A were extended to a range of other purine and pyrimidine nucleosides. All purine nucleosides that were tested, with the notable exception of tubercidin (7-deazaadenosine), totally inhibited adenosine transport in a monophasic manner. 2'-Deoxyinosine, 2'-deoxyadenosine, inosine, and guanosine were all potent inhibitors with apparent Ki values of less than 1 µM (Table I). To investigate further the kinetics of inhibition of adenosine influx, the effect of varying both the concentration of [3H]adenosine and the test nucleoside was examined. Fig. 3 shows the results of one such experiment with inosine where the data is plotted as 1/v versus I (Dixon plot). The plot is consistent with competitive inhibition and gave an apparent Ki value of 0.9 µM.

                              
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Table I
Rank order of inhibitors of adenosine influx by procyclic T. brucei brucei
Initial rates of 1 µM adenosine influx at 22 °C were determined as a function of varying concentrations of test compound and were plotted as a percent of control flux versus the concentration of the test compound as shown in Fig. 2. IC50 values were determined from the dose-response curves using the computer program Inplot. Apparent Ki values were calculated from the equation Ki = IC50/(1 + ([L]/Km)), where the Km value was taken as 0.26 µM and L = 1 µM adenosine. The values are the means ± S.E. of three separate experiments. NI, no inhibition, taken to be <10% decrease in adenosine transport at 100 µM test compound or 50 µM NBMPR.


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Fig. 3.   Dixon plot of adenosine influx inhibited by inosine. The reciprocals of adenosine uptake at 0.1 (bullet ), 0.25 (open circle ), 0.5 (black-square), and 1 (square ) µM are plotted against the respective concentrations of inosine. The value for the apparent Ki was 0.9 µM.

In contrast to the potent inhibition observed with purine nucleosides, pyrimidine nucleosides at concentrations as high as 100 µM failed to inhibit adenosine influx (Table I). Similarly, purine and pyrimidine nucleobases also had no effect on adenosine influx by T. brucei brucei. Nitrobenzylthioinosine (NBMPR) and dipyridamole, both inhibitors of the NBMPR-sensitive (es) mammalian facilitated-diffusion nucleoside carriers (13, 14, 19, 20), had a differential effect on adenosine influx by T. brucei brucei procyclics. NBMPR had no effect on transport up to concentrations of 50 µM, approximately 50,000 times the apparent Ki value for inhibition of the es nucleoside transporter by NBMPR (13, 14, 19, 20). Dipyridamole inhibited adenosine transport with a Ki of 0.64 ± 0.03 (n = 3) µM, a value several times higher than the effective concentration for inhibiting the mammalian es transporters but similar to the potency of dipyridamole to inhibit the NBMPR-insensitive (ei) nucleoside transporter in non-rat cells (13, 14, 19, 20).

Transport of [3H]Inosine-- As mentioned above, the Dixon plot of 1/(adenosine uptake rate) versus inosine concentration (Fig. 3) shows that inosine competitively inhibited adenosine transport with a Ki less than 1 µM. However, to demonstrate that inosine is actually transported by the adenosine carrier, the uptake of [3H]inosine in T. brucei brucei procyclics was studied. [3H]Inosine transport, at 50 nM, was rapid and linear for at least 60 s with a rate of 0.036 ± 0.003 pmol (107 cells)-1 s-1 (not shown). The concentration dependence of inosine influx was saturable and conformed to simple Michaelis-Menten kinetics (Km 0.36 ± 0.04 µM; Vmax 0.40 ± 0.02 pmol(107 cells)-1 s-1). Inosine uptake was inhibited by the purine nucleosides adenosine, guanosine, and 2'-deoxyinosine with Ki values of 0.15 ± 0.04, 0.21 ± 0.05, and 0.12 ± 0.02 µM, respectively (n = 3), but tubercidin had little effect except at high concentrations (>100 µM). Hill slopes for these inhibitors were all within 10% of -1, consistent with the presence of a single transporter. Purine bases (adenine), pyrimidine bases (uracil), and pyrimidine nucleosides (cytidine) had no effect on inosine uptake at concentrations up to 100 µM. Traces depicting the effects of adenosine, guanosine, tubercidin, and uracil on inosine transport are shown in Fig. 2B. These results demonstrate that inosine is transported by the same carrier as adenosine.

Mechanism-- Two major classes of mammalian transporters exist as follows: equilibrative facilitated-diffusion nucleoside transporters and Na+/nucleoside cotransporters (13, 14, 19, 20). To test whether adenosine uptake could be dependent on the sodium electrochemical gradient across the plasma membrane, time courses of adenosine transport were compared in the presence and absence of sodium (sodium replaced by N-methyl-D-glucamine). The initial rate of adenosine influx in the presence of N-methyl-D-glucamine was similar to that observed in the presence of sodium (92 ± 8% of the flux in the presence of Na+; mean ± S.E., n = 3) indicating a sodium-independent mechanism of transport.

In many prokaryotic organisms a proton electrochemical gradient across membranes is the driving force behind the uptake of a variety of nutrients (21-23). This gradient is composed of two components, the plasma membrane potential (Vm) and the pH gradient over the plasma membrane (Delta pH). To address whether a proton electrochemical gradient drives adenosine transport in T. brucei brucei procyclics, a series of experiments were conducted. In the first set of experiments, the proton gradient across the plasma membrane was modified by changing the extracellular pH (pHo), and the effects on adenosine transport were assessed. Consistent with a proton-driven system, a basic pHo significantly reduced adenosine uptake as compared with the control at pH 7.3 (36%, p < 0.001 at pH 8.0) (Fig. 4). However, an extracellular pH of 6.5 did not alter adenosine uptake rates (Table II). These effects of pHo on adenosine transport could be linked to similar effects on the protonmotive force (see Table II and below).


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Fig. 4.   Effect of extracellular pH on adenosine uptake by T. brucei brucei procyclics. Procyclic cells (25 µl) suspended in assay buffer (pH 7.3) were added to 175 µl of transport medium containing 1.14 µM [3H]adenosine at pH 7.3 (bullet ) and 8.0 (open circle ). Control experiments establish that at these ratios the test pH was unaffected by the addition of buffer at pH 7.3. The data points shown represent the mean of three determinations ± S.D. The rate of uptake at the two pH levels (0.50 ± 0.02 and 0.31 ± 0.01 pmol(107 cells)-1 s-1, respectively) was compared using a two-tailed t test and found to be significantly different (p < 0.001). Correlation coefficients exceeded 0.99 for both lines. The experiment was repeated on three separate occasions with different cell batches, and similar results were obtained.

                              
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Table II
Effects of ionophores, H+-ATPase inhibitors, and extracellular pH on adenosine transport and protonmotive force
Procyclic T. brucei brucei cells were preincubated under the indicated conditions, and the influx of 1 µM adenosine for 5 or 10 s was subsequently determined. Results are expressed as means ± S.E., n = 3-6. pHi and Vm were measured as described in the legends of Figs. 5 and 6, respectively, and in part derived from Ref. 10, allowing the protonmotive force (PMF) to be calculated from Delta pH and Vm. The extracellular pH (pHo) was 7.3 unless otherwise indicated. ND, not determined.

An additional method to study the ionic requirements for a transport process is with the use of ionophores with varying selectivity (Table II). The sodium ionophore monensin (5 µM) had no effect on transport (98 ± 3.5% of control after 15 min preincubation), confirming that sodium does not act as a potential driving force for adenosine influx in T. brucei brucei procyclics. However, the proton-gradient uncoupler CCCP dose-dependently reduced adenosine transport, reaching >75% inhibition at 20 µM. The concentration of CCCP required to inhibit 50% of the adenosine flux sensitive to CCCP was 2.5 ± 0.4 µM (data not shown). Similar reductions in adenosine transport were seen with the ionophore nigericin and the Na+/K+ exchanger gramicidin (Table II). In addition, two compounds reported to inhibit the T. brucei brucei plasma membrane H+-ATPase, N,N'-dicyclohexylcarbodiimide (DCCD) and N-ethylmaleimide (NEM) (24, 25), also inhibited adenosine uptake.

To verify that the above treatments were inhibiting adenosine influx by disrupting either the intracellular pH or the plasma membrane potential, the fluorescent dyes BCECF and bis-oxonol were used to determine pHi and Vm, respectively. Under resting conditions at 25 °C the pH of T. brucei brucei procyclics is 7.21 ± 0.03, and the Vm -93.6 ± 0.8 mV (10). Fig. 5 shows that CCCP (10 µM) and nigericin (20 µM), but not gramicidin (up to 10 µM), induced a sustained cytosolic acidification. In contrast, gramicidin (1 µM), as well as 10 µM CCCP, caused a marked plasma membrane depolarization, whereas up to 20 µM nigericin was almost without effect (Fig. 6). From these traces and similar experiments with other compounds, the changes in pHi and Vm were quantified, and the overall protonmotive force was calculated (Table II). Plotting the results listed in Table II as adenosine uptake versus protonmotive force (Fig. 7) yielded a linear relationship (r2 = 0.93). Under conditions where Delta pH or Vm were not constant (Table II), the correlation between the rate of adenosine uptake and Vm (r2 = 0.42) or Delta pH (r2 = 0.33) was much less than that shown between transport rates and PMF. However, transport of adenosine was dependent on Delta pH at near constant Vm (87.1 ± 1.9 or 75.3 ± 1.7 mV) and on Vm at near constant Delta pH (0.36 ± 0.05) (r2 = 0.94, 0.98, and 0.99, respectively), as predicted for an H+/adenosine cotransporter. These findings illustrate that inhibition of adenosine uptake does not require either cytosolic acidification or membrane depolarization per se but that either event, by reducing the protonmotive force, is sufficient to reduce adenosine uptake rates.


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Fig. 5.   Effects of ionophores on intracellular pH. BCECF-preloaded cells were suspended in assay buffer, and pHi was recorded fluorimetrically. At indicated times, CCCP (A, 10 µM), nigericin (B, 20 µM), or gramicidin (C, 10 µM) were added to the medium (solid lines). Dashed lines show control traces with the same volume of EtOH added. Effects of the ionophores were taken to be the average pHi level during the last 45 s of recording. Traces are representative of at least three similar experiments, and the average effect on pHi was used to calculate effects on the protonmotive force.


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Fig. 6.   Effects of ionophores on membrane potential. Procyclic cells (107) were equilibrated with 0.1 µM bis-oxonol in assay buffer, and fluorescence intensity at 580 nm was recorded (excitation at 540 nm). Traces shown are after background subtraction (trace in the absence of cells). At 60 s, either ionophore (A, 10 µM CCCP; B, 20 µM nigericin; C, 1 µM gramicidin; solid lines) or the same volume of solvent (EtOH, dashed lines) was added, and fluorescence intensity was recorded for a further 3 min. Traces are representative of at least three similar experiments, and the average effect on Vm, taken as the average potential during the last minute of recording (gramicidin 30 s), was used to calculate effects on the protonmotive force.


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Fig. 7.   Correlation between adenosine uptake and protonmotive force. The effects of ionophores and variations of pHo on adenosine uptake (Table II) was plotted against their effects on the PMF. The line was calculated by linear regression (r2 = 0.93); error bars are S.E.

The effects of the treatments listed in Table II on cellular ATP were also determined to test whether these treatments could be linked to a general reduction in ATP levels and hence in active transport. Nigericin (20 µM) and CCCP (10 µM) did reduce ATP levels by 72 and 30%, respectively, but 1 µM gramicidin and 1 mM NEM had no significant effect on the intracellular ATP concentration. However, all four treatments had a similar inhibitory effect on adenosine transport. Moreover, 10 µM DCCD, which had no effect on adenosine uptake following a 3-min preincubation period, reduced cellular ATP content by 33% over the same time interval. Thus, there was no correlation between ATP content and the rate of adenosine influx.

To ascertain whether direct proton influx is associated with adenosine uptake, the effect of adenosine on the intracellular pH of procyclic T. brucei brucei was investigated. Addition of adenosine (10 µM) to procyclic cells in the standard assay buffer did not detectably alter pHi (data not shown). However, in an attempt to improve the detection of the possible co-flux of protons with adenosine, BCECF-loaded procyclic cells were "base-loaded" by the addition of 20 mM NH4Cl. This treatment produced a rapid rise in pHi resulting in a more alkaline base line within the cells. By using these base-loaded cells, the effect of adding either buffer or buffer containing 10 µM adenosine on the rate with which the cells recovered their pre-NH4Cl pHi was compared. Fig. 8 shows that compared with buffer alone, addition of adenosine accelerated the rate of recovery in pHi toward that found prior to the addition of ammonium. The average of eight separate experiments revealed that after 3 min a 38 ± 4% recovery to original base-line pHi in the presence of adenosine was demonstrable compared with less then half that recovery (17 ± 4%, n = 7; p < 0.01) seen when buffer alone was added. This result is consistent with the cotransport of protons with adenosine.


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Fig. 8.   Adenosine enhances the recovery of pHi in base-loaded procyclic T. brucei brucei. Procyclic cells were base-loaded at the indicated time by the addition of 30 µl of 2 M NH4Cl to a cuvette containing approx 107 BCECF-loaded cells in 3 ml of assay buffer. Once a new stable level of pHi was established, adenosine (final concentration 25 µM, dotted line) or an equal volume of assay buffer (solid line) was added at the indicated time. Data shown are the average of eight traces in the presence of adenosine and seven control traces. Steady state levels of pHi after adenosine treatment (n = 8) were taken as the average pH during the last 60 s of recording and were significantly different from control (n = 7) by unpaired Student t test (7.61 ± 0.048 and 7.77 ± 0.056%, respectively, p < 0.01).

This hypothesis also requires that uptake of (neutral) adenosine be electrogenic. Addition of 10 µM adenosine did not change the membrane potential of procyclic T. brucei brucei as monitored with bis-oxonol (data not shown). Like the absence of a direct effect of adenosine on pHi, this might be due to compensation of the proton influx by a plasma membrane H+-ATPase. To test this possibility, procyclic cells were treated with 1 mM NEM, which inhibits the T. brucei brucei proton pump (24). NEM induced a gradual acidification of the procyclic cytosol (Fig. 9A), as well as a slow plasma membrane depolarization (Fig. 9B), consistent with a slow influx of protons. Subsequent addition of 20 µM of either adenosine (Fig. 9B) or 2-chloroadenosine (not shown) induced a significantly greater depolarization than controls receiving assay buffer (Vm = -48.8 ± 2.1, p < 0.05, -44.1 ± 0.8, p < 0.01, and -57.2 ± 0.9 mV, respectively).


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Fig. 9.   Effects of N-ethylmaleimide on intracellular pH and adenosine-induced membrane depolarization in procyclic T. brucei brucei. A, effect of 1 mM NEM on pHi. Traces were recorded as described in the legend to Fig. 5. NEM (1 mM, solid line) or the same volume of assay buffer (dashed line) was added at 50 s. Traces shown are representative of four different experiments with different batches of cells. B, effect of adenosine on Vm after NEM treatment. Vm was recorded as described in the legend to Fig. 6. NEM (1 mM) was added at 60 s, and after an additional 2 min, 30 µl of either adenosine (final concentration 40 µM; solid line) or assay buffer (dashed line) was added, and fluorescence intensity was recorded for 5 more min. Steady-state levels of membrane potential were taken as the average Vm over the last 2 min. Traces shown are representative of three similar experiments.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Analysis of the transporter-mediated entry of metabolized permeants requires methods that will allow measurements of the initial rates of influx. Requirements for such methods are definitive time courses of permeant uptake which need both a determination of zero time origins of such time courses and a rapid means of separating cells from the extracellular isotope. In the present study we have used excess unlabeled permeant to block influx of labeled permeant together with centrifuging the cells through an oil layer to obtain initial rates of influx (Fig. 1). The results of Fig. 1 also suggest that the time required for T. brucei brucei procyclics to centrifuge through the oil layer is 4 s, considerably longer than the 2 s estimated for mammalian cells (14, 15). Moreover, during this centrifugation period adenosine influx appears to continue if unlabeled excess permeant has not been added. A similar delay of 6 s in centrifuging cells below the oil has also recently been reported for the protozoan parasite, Giardia lamblia (26). Taken together these results indicate that it is inappropriate to assume a lag time of 2 s for centrifuging protozoan parasites through the oil layer and that individual estimates should be made for each cell type.

By using the above methods, the results presented here demonstrate that a single saturable mechanism is responsible for the inward transport of adenosine by cultured procyclic forms of T. brucei brucei. The apparent affinity (Km) for the transport of adenosine at 22 °C is submicromolar (0.26 ± 0.02 µM) and on average 1 to 2 orders of magnitude higher than values obtained for mammalian nucleoside transporters (9, 15, 19, 20). As such, this should enable the parasite to effectively scavenge the purine nucleoside. The specificity of this transporter appears to be solely toward purine nucleosides, with the naturally occurring nucleosides inosine and guanosine being potent inhibitors of adenosine influx. Interestingly, the adenosine analogue 8-azidoadenosine showed a large increase (at least 20-fold) in its apparent Ki value (5.2 ± 0.07 µM) compared with the affinity constant for adenosine. In addition tubercidin, another adenosine analogue, almost completely failed to inhibit adenosine influx. These results suggest that specific positions on the purine ring are important determinants for the permeant specificity of the transporter in T. brucei brucei procyclics. The fact that [3H]inosine was taken up with a Km similar to the Ki value for inosine on adenosine transport and that inosine transported was inhibited in turn by adenosine with a Ki value identical to the Km for adenosine strongly suggests that inosine and adenosine are taken up by the same transporter. This idea is further supported by the identical actions of inhibitors ranging from pyrimidine nucleobases to purine nucleoside analogues and by the observation that the slopes of inhibitor plots was always near -1, both for adenosine and inosine uptake. By extension, it is possible to speculate that this transporter mediates the uptake of all natural purine nucleosides with high affinity, but a definite answer awaits separate studies with each radiolabeled nucleoside.

The strict selectivity for purine nucleosides observed for the adenosine transporter in T. brucei brucei procyclics is unusual. Related parasites, such as Leishmania donovani promastigotes and the insect trypanosomatid Crithidia lucillae, possess an adenosine carrier which is inhibited by pyrimidine nucleosides (16, 27). Nevertheless, a single dipyridamole-sensitive adenosine transporter has been suggested to be present in Toxoplasma gondii tachyzoites (28), showing a specificity for purines only. We recently reported the existence of a purine-selective nucleobase transporter in T. brucei brucei procyclics (10, 29). These transporters together appear to provide T. brucei brucei procyclics with high affinity uptake capacity for all natural purines. Neither transporter exhibits any affinity for pyrimidines, which T. brucei brucei can synthesize de novo (1).

The presence of a single adenosine transporter in T. brucei brucei procyclics contrasts markedly with the two adenosine transport systems (P1 and P2) in bloodstream forms of the parasite (5). Interestingly, the Km and Vmax for the procyclic adenosine transporter are similar to that reported for the P1 system in bloodstream forms. In addition, from the limited inhibition data presented on the P1 transporter in bloodstream trypanosomes of T. brucei (5),2 its apparent permeant selectivity is identical to that determined in this study for the adenosine transporter in procyclics. It is therefore possible that this transporter (P1) is constitutively expressed in both forms of the parasite, whereas an additional transporter (P2) is expressed in bloodstream stages alone. Interestingly, a similar situation appears to exist with respect to purine nucleobase transporters in T. brucei brucei. Bloodstream forms of T. brucei brucei express at least two purine nucleobase transporters in addition to the P1 and P2 nucleoside transporters (30). It has been suggested that environmental factors, such as temperature and pH, are important for transformation into different life cycle stages for both Trypanosoma cruzi (31) and L. donovani (32). Similar factors may also trigger expression of membrane transporters.

The mechanism of nucleoside transport in parasitic protozoa has received little attention. The current study has demonstrated that adenosine influx in T. brucei brucei procyclics is an energy-dependent process as exemplified by the inhibitory effects of the ionophores gramicidin, CCCP, and nigericin. The energy source was not the transmembrane sodium gradient as neither monensin, a Na+ ionophore, nor replacement of Na+ with NMG+ had any effect on adenosine transport rates. Similarly, the lack of correlation between cellular ATP content and the effects of the ionophores NEM and DCCD on adenosine influx demonstrates that adenosine uptake was not directly coupled to the cytosolic ATP concentration. However, a range of treatments that lead to a dissipation of the proton electrochemical gradient across the cell membrane, either through cytosolic acidification (nigericin), membrane depolarization (gramicidin), or both (CCCP), inhibited adenosine transport. This finding indicates that adenosine transport is coupled to the protonmotive force, which consists of both Delta pH and Vm components, and Fig. 7 demonstrates a linear correlation between protonmotive force and adenosine transport in T. brucei brucei procyclics.

The simplest model to explain the above findings is that the procyclic adenosine transporter functions as an H+ symporter. Evidence to support this mechanism comes from both direct and indirect approaches and includes the following. (i) Changing the external pH from 7.3 to 8.0 inhibited adenosine transport. Inhibition was associated with a marked effect on the protonmotive force (Table II). Unexpectedly, acidification of the medium failed to stimulate adenosine transport. However, the present data (Table II) show that at pHo = 6.4, the pH gradient across the procyclic membrane is near zero, and the protonmotive force is similar to that observed at pHo = 7.3. (ii) The influx of adenosine should be accompanied by an electrogenic proton influx if the carrier is functioning as an adenosine/proton symporter. Direct measurements of pHi failed to confirm these properties because T. brucei brucei procyclics maintain their internal pH very efficiently near neutral (10, 33) with the help of a plasma membrane H+-ATPase. However, base loading with NH4Cl did allow demonstration of an adenosine-induced proton influx (Fig. 8), as the alkaline cytosolic pH effectively prevents the action of the proton pump. (iii) The electrogenic nature of adenosine influx was demonstrated after pretreating the procyclic cells with NEM, a procedure that will inhibit the plasma membrane H+-ATPase (34). Under these conditions, adenosine induced a modest but significant depolarization (Fig. 9). It is unlikely that this depolarization was due to the metabolism of adenosine (generating ATP), as a similar depolarization was demonstrated with 2-chloroadenosine, an adenosine analogue that is not deaminated and is only weakly phosphorylated (35, 36).

Nucleoside analogues have been used in pilot studies for anti-leishmanial (37) and anti-schistosomal (38) regimes, the latter in combination with NBMPR to facilitate host cell protection by inhibiting mammalian transporters. Results presented here indicate differences exist between mammalian and trypanosome procyclic nucleoside transporters. In addition, the results overwhelmingly support the conclusion that the T. brucei brucei procyclics use the proton rather than the sodium gradient to transport purine nucleosides. If further characterization of bloodstream forms of the parasite reveals similar differences, the chances of developing new chemotherapeutic protocols by using, for example, a cytotoxic nucleoside analogue selective for the parasite transporter(s) would seem hopeful.

    FOOTNOTES

* This work was supported by The Wellcome Trust and partly undertaken within the Wellcome Trust funded Protein Science Facility.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of a BBSRC graduate studentship award.

§ To whom correspondence should be addressed: Research School of Biosciences, The University, Canterbury, Kent CT2 7NJ, UK. Tel.: 01227-827581; Fax: 01227-763912; E-mail: S.M.Jarvis{at}ukc.ac.uk.

1 The abbreviations used are: BCECF/AM, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester; bis-oxonol, bis-(1,3-diethylthiobarbituric acid)trimethine oxonol; pHo, extracellular pH; pHi, intracellular pH; CCCP, carbonyl cyanide chlorophenylhydrazone; DCCD, N,N'-dicyclohexylcarbodiimide; NBMPR, nitrobenzylthioinosine; Mops, 3-(N-morpholino)propanesulfonic acid; Mes, 4-morpholineethanesulfonic acid; NEM, N-ethylmaleimide; Vm, membrane potential; PMF, protonmotive force; NMG+, N-methyl-D-glucamine.

2 H. P. de Koning and S. M. Jarvis, unpublished results.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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