From the Research School of Biosciences, University of Kent,
Canterbury, Kent CT2 7NJ, United Kingdom
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INTRODUCTION |
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.
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EXPERIMENTAL PROCEDURES |
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 (
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).
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(Eq. 1)
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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.
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RESULTS |
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 ( ) or by addition of 1 ml of assay buffer
containing 4 mM adenosine followed by immediate
centrifugation ( ). 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.).
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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 ( ), adenosine ( ), and guanosine
( ). 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 ( ), guanosine ( ), tubercidin ( ), and
uracil ( ). 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.
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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 ( ), 0.25 ( ), 0.5 ( ), and 1 ( ) µM are plotted against the
respective concentrations of inosine. The value for the apparent
Ki was 0.9 µM.
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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 (
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 ( ) and 8.0 ( ). 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 pH and
Vm. The extracellular pH (pHo) was 7.3 unless otherwise indicated. ND, not determined.
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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
pH or Vm were not constant (Table II), the
correlation between the rate of adenosine uptake and
Vm (r2 = 0.42) or
pH
(r2 = 0.33) was much less than that shown
between transport rates and PMF. However, transport of adenosine was
dependent on
pH at near constant Vm (87.1 ± 1.9 or 75.3 ± 1.7 mV) and on Vm at near
constant
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.
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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
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).
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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.
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DISCUSSION |
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
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.