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

Abstract

Adenosine transport at 22 degrees 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 microM, Vmax 0.63 +/- 0.18 pmol/10(7) cells s-1). Purine nucleosides, with the exception of tubercidin (7-deazaadenosine), and dipyridamole inhibited adenosine influx (Ki 0.18-5.2 microM). 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.

Full text

Characterization of a Nucleoside/Proton Symporter in Procyclic
Trypanosoma brucei brucei*
(Received for publication, July 2, 1997, and in revised form, December 3, 1997)
Harry P. de Koning, Christopher J. Watson‡, and Simon M. Jarvis§
From the Research School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, United Kingdom
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
(K
m
0.26 60.02
m
M,V
max
0.63 60.18 pmol/10
7
cells s
21
).
Purine nucleosides, with the exception of tubercidin
(7-deazaadenosine), and dipyridamole inhibited adeno-
sine influx (K
i
0.18 –5.2
m
M). Purine nucleobases and py-
rimidine 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 trypano-
somes. Uptake of adenosine was Na
1
-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-car-
boxyfluorescein acetoxymethyl ester, respectively) in-
hibited adenosine transport. Similarly, an increase in
extracellular pH from 7.3 to 8.0 reduced adenosine in-
flux by 30%. A linear correlation was demonstrated be-
tween the rate of adenosine transport and the proton-
motive 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
1
symporter.
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, in-
cluding 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 car-
ried out (for review see Ref. 1), the mode by which purines are
taken up from the environment by T. brucei has received lim-
ited attention. The first report on the uptake of nucleosides by
T. brucei appeared in 1980 (4). Adenosine uptake was sug-
gested to occur in a manner consistent with two mechanisms,
one with high affinity and the other with low affinity for aden-
osine. 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 K
m
values for adenosine influx of 0.15 and 0.59
m
M, respectively (5).
The P1 system was selectively inhibited by inosine, whereas
the P2 transporter was specifically blocked by adenine. Inter-
estingly, one of these carriers (P2) appeared to transport mel-
amino-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 func-
tional 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 pres-
ent 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
Trypanosome Culture—Procyclic forms of T. brucei brucei strain 427
were grown as described by Brun and Scho¨nenberger (8) in SDM-79
medium. Cells at mid-logarithmic stage of growth were harvested and
washed twice in assay buffer (33 mMHepes, 98 mMNaCl, 4.6 mMKCl,
0.3 mMCaCl
2
, 0.07 mMMgSO
4
, 5.8 mMNaH
2
PO
4
, 0.3 mMMgCl
2
,23mM
NaHCO
3
and 14 mMglucose, pH 7.3). Cells were resuspended in buffer
to approximately 10
8
cells/ml.
Nucleoside Transport Measurements—All measurements and dilu-
tions 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 per-
meant. 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
m
l of oil upon which was layered 100
m
l of transport
medium containing
3
H-nucleoside (10
m
Ci/ml). Transport was initiated
* This work was supported by The Wellcome Trust and partly under-
taken 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. This article must therefore be hereby marked “adver-
tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
‡ 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@ukc.ac.uk.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 16, Issue of April 17, pp. 9486–9494, 1998
© 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org9486

by addition of 100
m
l of procyclic T. brucei brucei cells ('10
7
). 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 mMunlabeled permeant), followed
by immediate centrifugation for 30 s at 12,000 3g. 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 scin-
tillation 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
3
H-permeant and 4 mMunlabeled permeant. Where applicable, ki-
netic constants of influx (apparent K
m
and V
max
) and inhibition con-
stants (IC
50
values and apparent K
i
) were determined by nonlinear
regression analysis of data using appropriate equations and the com-
puter programs Enzfitter (Elsevier Biosoft) and Inplot (Graphpad
Software).
In inhibition studies, test compounds and [
3
H]adenosine or
[
3
H]inosine were added to cells simultaneously, except metabolic inhib-
itors and ionophores that were preincubated with cells for 1–10 min
before addition of [
3
H]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 ex-
pressed per 10
7
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 [
3
H]H
2
O with either [
14
C]sucrose or [
14
C]mannitol as the extracellu-
lar 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 K
m
and K
i
values are given 6S.E. unless
otherwise indicated.
Metabolism—To explore the metabolic fate of transported substrate,
the inert oil layer was underlayered with 100
m
l of 20% (w/v) perchloric
acid. The assay was performed as described for the transport experi-
ments, 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 MKOH. Precipitate was removed by centrifugation (2 min,
12,000 3g), 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 (R
f
values of 0, 0.53, 0.63, 0.46, and
0.36 for ATP, adenosine, adenine, hypoxanthine, and inosine, respec-
tively). 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
m
MBCECF/
AM, and incubated at 25 °C for1hatadensity of approximately 10
8
cells/ml. Cells were pelleted and washed twice with assay buffer to
remove extracellular dye and resuspended at a final cell density of 10
8
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 (pH
i
)versus fluorescence was carried out
using 20
m
Mnigericin in a K
1
-rich Ringers solution (20 mMKCl, 10 mM
NaCl, 2 mMMgCl
2
, 100 mMpotassium gluconate, 10 mMglucose, 5 mM
Mes, and 5 mMMops) 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 (V
m
) was measured and calibrated using the fluorescent ani-
onic probe bis-oxonol as described (10), with excitation at 540 nm and
emission recorded at 580 nm. Briefly, 100
m
l of procyclic cells (10
7
)or
assay buffer were added to 2.9 ml of 0.1
m
Mbis-oxonol in assay buffer in
a cuvette in the fluorimeter. Fluorescence was recorded, and the V
m
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).
PMF 5Vm2~2.3RT/F!~pHi2pHo!(Eq. 1)
in which pH
o
is the extracellular pH.
ATP Measurements—Intracellular measurements of ATP were de-
termined using a bioluminescent assay, as described previously (10).
Materials—Nucleosides, nucleobases, and ionophores were pur-
chased from Sigma. [2,8,59-
3
H]Adenosine (2.3 TBq/mmol) was obtained
from NEN Life Science Products. [
3
H]Inosine (1.4 TBq/mmol) was pur-
chased from Moravek Biochemicals. [
3
H]H
2
O (925 MBq/g), [
14
C]sucrose
(23.3 GBq/mmol), D-[1-
14
C]mannitol (2.11 GBq/mmol), and [
3
H]thymi-
dine (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
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 tech-
nique relies on the termination of uptake by simply centrifug-
ing 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 vari-
ety of laboratories to determine initial transport rates of vari-
ous 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 mMadenosine, followed immediately by centrifuga-
tion 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. How-
ever, the intercept on the ordinate for the two time courses was
markedly different. In the presence of 4 mMadenosine, the time
course intercepted with the estimate of extracellular space.
Without the addition of unlabeled adenosine, [
3
H]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 24 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 per-
meant was used to terminate transport.
Metabolism of Permeant—Metabolism of 1
m
M[
3
H]adenosine
was monitored by TLC following an incubation period of 0, 5,
and 10 s. After5s7%oftherecovered 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 60.02
m
l per 10
7
cells; S.D., n53), we calculate that the
1
The abbreviations used are: BCECF/AM, 29,79-bis(carboxyethyl)-
5,6-carboxyfluorescein acetoxymethyl ester; bis-oxonol, bis-(1,3-diethyl-
thiobarbituric acid)trimethine oxonol; pH
o
, extracellular pH; pH
i
, intra-
cellular pH; CCCP, carbonyl cyanide chlorophenylhydrazone; DCCD,
N,N9-dicyclohexylcarbodiimide; NBMPR, nitrobenzylthioinosine; Mops,
3-(N-morpholino)propanesulfonic acid; Mes, 4-morpholineethanesulfo-
nic acid; NEM, N-ethylmaleimide; V
m
, membrane potential; PMF, pro-
tonmotive force; NMG
1
,N-methyl-D-glucamine.
Adenosine Transport in Trypanosomes 9487

small amount of unmetabolized adenosine present did not ex-
ceed the concentration of that found extracellularly. The prac-
tical 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. 1Aand Ref. 18).
Kinetic Parameters of Adenosine Transport—Initial rates of
adenosine transport were measured over the concentration
range 0.05–5
m
Mfor [
3
H]adenosine. Fig. 1Bshows that adeno-
sine influx was saturable and conformed to simple Michaelis-
Menten kinetics (K
m
0.19 60.04
m
M;V
max
0.45 60.02 pmol/10
7
cells s
21
). The mean values from three separate experiments
were 0.26 60.02
m
Mand 0.63 60.18 pmol/10
7
cells s
21
for K
m
and V
max
, respectively.
Substrate Specificity of the Adenosine Transporter—The sub-
strate 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 [
3
H]adenosine influx in a monopha-
sic manner with Hill slopes of 1.2 60.4 and 0.94 60.1, respec-
tively. These findings are consistent with the suggestion that
both nucleosides are permeants for the same carrier. The ap-
parent K
i
value derived from this data for self-inhibition of
adenosine influx was 0.25 60.05
m
M(n53), a value similar to
the apparent K
m
estimate. In contrast, thymidine had no sig-
nificant effect on adenosine influx by T. brucei brucei procyclics
at concentrations up to 100
m
M. In a parallel experiment, the
flux of 1
m
M[
3
H]thymidine (0.014 pmol/10
7
cells s
21
) was de-
termined to be 50-fold less than the corresponding rate of
adenosine transport at the same concentration.
The studies shown in Fig. 2Awere extended to a range of
other purine and pyrimidine nucleosides. All purine nucleo-
sides that were tested, with the notable exception of tubercidin
(7-deazaadenosine), totally inhibited adenosine transport in a
monophasic manner. 29-Deoxyinosine, 29-deoxyadenosine, in-
osine, and guanosine were all potent inhibitors with apparent
K
i
values of less than 1
m
M(Table I). To investigate further the
kinetics of inhibition of adenosine influx, the effect of varying
both the concentration of [
3
H]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 K
i
value of 0.9
m
M.
In contrast to the potent inhibition observed with purine
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
m
l of procyclic cells (10
7
cells) to 100
m
l of transport
medium containing 2
m
M[
3
H]adenosine. After the appropriate time
interval, transport was terminated by either centrifugation alone (M)or
by addition of 1 ml of assay buffer containing 4 mMadenosine followed
by immediate centrifugation (f). B, concentration dependence of aden-
osine influx. Procyclic cells were incubated with graded concentrations
(0.1–5
m
M)of[
3
H]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 concen-
tration. The kinetic constants were determined by nonlinear regression
analysis using the Michaelis-Menten equation and gave a K
m
value of
0.19 60.04
m
Mand a V
max
of 0.45 60.02 pmol (10
7
cells)
21
s
21
(6S.E.).
FIG.2.Inhibition of adenosine and
inosine transport in T. brucei brucei
procyclics. A, uptake of 1
m
M[
3
H]ade-
nosine at 5 s was determined in the pres-
ence of thymidine (), adenosine (f), and
guanosine (E). The results are the aver-
age of three separate experiments (6
S.E.) and are presented as a percentage of
the control influx rate (0.7 pmol (10
7
cells)
21
s
21
) (see Table I for K
i
values). B,
uptake of 50 nM[
3
H]inosine at 10 s was
determined in the presence of adenosine
(f), guanosine (E), tubercidin (‚), and
uracil (Œ). The results are the average of
triplicate determinations (6S.E.) and are
representative of three similar experi-
ments. K
i
values for the adenosine and
guanosine traces shown were 0.20 6
0.003 and 0.28 60.02
m
M, respectively.
Adenosine Transport in Trypanosomes9488

nucleosides, pyrimidine nucleosides at concentrations as high
as 100
m
Mfailed to inhibit adenosine influx (Table I). Similarly,
purine and pyrimidine nucleobases also had no effect on aden-
osine influx by T. brucei brucei. Nitrobenzylthioinosine
(NBMPR) and dipyridamole, both inhibitors of the NBMPR-
sensitive (es) mammalian facilitated-diffusion nucleoside car-
riers (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
m
M, approximately 50,000
times the apparent K
i
value for inhibition of the es nucleoside
transporter by NBMPR (13, 14, 19, 20). Dipyridamole inhibited
adenosine transport with a K
i
of 0.64 60.03 (n53)
m
M, a value
several times higher than the effective concentration for inhib-
iting the mammalian es transporters but similar to the potency
of dipyridamole to inhibit the NBMPR-insensitive (ei) nucleo-
side transporter in non-rat cells (13, 14, 19, 20).
Transport of [
3
H]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 K
i
less than 1
m
M. However, to demonstrate
that inosine is actually transported by the adenosine carrier,
the uptake of [
3
H]inosine in T. brucei brucei procyclics was
studied. [
3
H]Inosine transport, at 50 nM, was rapid and linear
for at least 60 s with a rate of 0.036 60.003 pmol (10
7
cells)
21
s
21
(not shown). The concentration dependence of inosine in-
flux was saturable and conformed to simple Michaelis-Menten
kinetics (K
m
0.36 60.04
m
M;V
max
0.40 60.02 pmol(10
7
cells)
21
s
21
). Inosine uptake was inhibited by the purine nucleosides
adenosine, guanosine, and 29-deoxyinosine with K
i
values of
0.15 60.04, 0.21 60.05, and 0.12 60.02
m
M, respectively (n5
3), but tubercidin had little effect except at high concentrations
(.100
m
M). Hill slopes for these inhibitors were all within 10%
of 21, consistent with the presence of a single transporter.
Purine bases (adenine), pyrimidine bases (uracil), and pyrimi-
dine nucleosides (cytidine) had no effect on inosine uptake at
concentrations up to 100
m
M. Traces depicting the effects of
adenosine, guanosine, tubercidin, and uracil on inosine trans-
port 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
1
/nucleoside cotransporters (13, 14, 19,
20). To test whether adenosine uptake could be dependent on
the sodium electrochemical gradient across the plasma mem-
brane, 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 68% of the flux in the presence
of Na
1
; mean 6S.E., n53) indicating a sodium-independent
mechanism of transport.
In many prokaryotic organisms a proton electrochemical gra-
dient 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 (V
m
) and the
pH gradient over the plasma membrane (DpH). 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 chang-
ing the extracellular pH (pH
o
), and the effects on adenosine
transport were assessed. Consistent with a proton-driven sys-
tem, a basic pH
o
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 pH
o
on
adenosine transport could be linked to similar effects on the
protonmotive force (see Table II and below).
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
m
M)
had no effect on transport (98 63.5% of control after 15 min
preincubation), confirming that sodium does not act as a po-
tential driving force for adenosine influx in T. brucei brucei
procyclics. However, the proton-gradient uncoupler CCCP dose-
dependently reduced adenosine transport, reaching .75% in-
hibition at 20
m
M. The concentration of CCCP required to
inhibit 50% of the adenosine flux sensitive to CCCP was 2.5 6
0.4
m
M(data not shown). Similar reductions in adenosine trans-
port were seen with the ionophore nigericin and the Na
1
/K
1
exchanger gramicidin (Table II). In addition, two compounds
reported to inhibit the T. brucei brucei plasma membrane H
1
-
ATPase, N,N9-dicyclohexylcarbodiimide (DCCD) and N-ethyl-
maleimide (NEM) (24, 25), also inhibited adenosine uptake.
To verify that the above treatments were inhibiting adeno-
TABLE I
Rank order of inhibitors of adenosine influx by procyclic T. brucei
brucei
Initial rates of 1
m
Madenosine 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. IC
50
values were determined from the dose-response
curves using the computer program Inplot. Apparent K
i
values were
calculated from the equation K
i
5IC
50
/(1 1([L]/K
m
)), where the K
m
value was taken as 0.26
m
Mand L 51
m
Madenosine. The values are the
means 6S.E. of three separate experiments. NI, no inhibition, taken to
be ,10% decrease in adenosine transport at 100
m
Mtest compound or
50
m
MNBMPR.
Compound K
i
(
m
M)6S.E.
29-Deoxyinosine 0.18 60.07
Adenosine 0.25 60.05
29-Deoxyadenosine 0.37 60.15
Dipyridamole 0.64 60.03
Inosine 0.72 60.11
Guanosine 0.94 60.06
2-Chloroadenosine 1.1 60.02
Formycin B 1.1 60.17
8-Azidoadenosine 5.2 60.07
Tubercidin .100
Adenine NI
Cytidine NI
Hypoxanthine NI
Thymidine NI
Uracil NI
Uridine NI
NBMPR NI
FIG.3.Dixon plot of adenosine influx inhibited by inosine. The
reciprocals of adenosine uptake at 0.1 (●), 0.25 (E), 0.5 (f), and 1 (M)
m
M
are plotted against the respective concentrations of inosine. The value
for the apparent K
i
was 0.9
m
M.
Adenosine Transport in Trypanosomes 9489

sine influx by disrupting either the intracellular pH or the
plasma membrane potential, the fluorescent dyes BCECF and
bis-oxonol were used to determine pH
i
and V
m
, respectively.
Under resting conditions at 25 °C the pH of T. brucei brucei
procyclics is 7.21 60.03, and the V
m
293.6 60.8 mV (10). Fig.
5 shows that CCCP (10
m
M) and nigericin (20
m
M), but not
gramicidin (up to 10
m
M), induced a sustained cytosolic acidifi-
cation. In contrast, gramicidin (1
m
M), as well as 10
m
MCCCP,
caused a marked plasma membrane depolarization, whereas
up to 20
m
Mnigericin was almost without effect (Fig. 6). From
these traces and similar experiments with other compounds,
the changes in pH
i
and V
m
were quantified, and the overall
protonmotive force was calculated (Table II). Plotting the re-
sults listed in Table II as adenosine uptake versus protonmo-
tive force (Fig. 7) yielded a linear relationship (r
2
50.93).
Under conditions where DpH or V
m
were not constant (Table
II), the correlation between the rate of adenosine uptake and
V
m
(r
2
50.42) or DpH (r
2
50.33) was much less than that
shown between transport rates and PMF. However, transport
of adenosine was dependent on DpH at near constant V
m
(87.1 61.9 or 75.3 61.7 mV) and on V
m
at near constant DpH
(0.36 60.05) (r
2
50.94, 0.98, and 0.99, respectively), as pre-
dicted for an H
1
/adenosine cotransporter. These findings illus-
trate that inhibition of adenosine uptake does not require ei-
ther cytosolic acidification or membrane depolarization per se
but that either event, by reducing the protonmotive force, is
sufficient to reduce adenosine uptake rates.
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
m
M) and CCCP (10
m
M) did
reduce ATP levels by 72 and 30%, respectively, but 1
m
Mgram-
icidin and 1 mMNEM had no significant effect on the intracel-
lular ATP concentration. However, all four treatments had a
similar inhibitory effect on adenosine transport. Moreover, 10
m
MDCCD, 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 correla-
tion 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
m
M) to procyclic cells in the standard assay buffer
did not detectably alter pH
i
(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 mMNH
4
Cl. This treatment
produced a rapid rise in pH
i
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
m
Madenosine on
the rate with which the cells recovered their pre-NH
4
Cl pH
i
was compared. Fig. 8 shows that compared with buffer alone,
addition of adenosine accelerated the rate of recovery in pH
i
toward that found prior to the addition of ammonium. The
average of eight separate experiments revealed that after 3
mina3864% recovery to original base-line pH
i
in the pres-
ence of adenosine was demonstrable compared with less then
half that recovery (17 64%, n57; p,0.01) seen when buffer
alone was added. This result is consistent with the cotransport
of protons with adenosine.
This hypothesis also requires that uptake of (neutral) aden-
FIG.4.Effect of extracellular pH on adenosine uptake by T.
brucei brucei procyclics. Procyclic cells (25
m
l) suspended in assay
buffer (pH 7.3) were added to 175
m
l of transport medium containing
1.14
m
M[
3
H]adenosine at pH 7.3 (●) and 8.0 (E). 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 6S.D. The rate of uptake at the two pH levels (0.50 6
0.02 and 0.31 60.01 pmol(10
7
cells)
21
s
21
, respectively) was compared
using a two-tailed ttest and found to be significantly different (p,
0.001). Correlation coefficients exceeded 0.99 for both lines. The exper-
iment was repeated on three separate occasions with different cell
batches, and similar results were obtained.
TABLE II
Effects of ionophores, H
1
-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
m
Madenosine for 5 or 10 s was subsequently
determined. Results are expressed as means 6S.E., n53–6. pH
i
and V
m
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 DpH and V
m
. The extracellular pH (pH
o
) was 7.3
unless otherwise indicated. ND, not determined.
Inhibitor or pH
o
PI Transport pH
i
V
m
PMF
min % control mV mV
None 100 7.21 60.03 286.4 61.3 280.8 62.2
CCCP (5
m
M)35864.2 6.93 60.04 257.8 63.9 236.0 64.2
CCCP (10
m
M)34163.5 6.86 60.06 248.9 61.8 222.8 63.7
Nigericin (5
m
M)38367.5 7.08 60.02 277.2 64.7 264.2 64.9
Nigericin (10
m
M)37065.0 6.80 60.05 270.0 63.7 240.5 64.8
Nigericin (20
m
M)33864.7 6.46 60.08 278.8 60.7 229.1 64.8
Gramicidin (1
m
M)347613 7.33 60.04 218.3 65.1 220.1 65.6
1
NEM (1 mM)34065.6 6.48 60.01 275.0 63.5 226.8 63.6
DCCD (10
m
M)39661.8 7.02 60.04 291.0 62.1 273.4 63.2
DCCD (100
m
M)34363.6 6.20 60.04 260.0 67.9 219.8 68.2
pH
o
56.5 5 94 62.1 6.40 60.03 289.8 60.5 283.1 61.8
pH
o
58.0 5 64 61.6 7.12 60.03 281.3 63.0 229.4 63.4
1
The effect of gramicidin on V
m
may not have reached steady state levels at 3 min. However, the value given for PMF does reflect the conditions
for adenosine uptake after the indicated 3 min preincubation. PI, preincubation time.
Adenosine Transport in Trypanosomes9490

osine be electrogenic. Addition of 10
m
Madenosine 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 pH
i
, this might be due to com-
pensation of the proton influx by a plasma membrane H
1
-
ATPase. To test this possibility, procyclic cells were treated
with 1 mMNEM, which inhibits the T. brucei brucei proton
pump (24). NEM induced a gradual acidification of the procy-
clic cytosol (Fig. 9A), as well as a slow plasma membrane
depolarization (Fig. 9B), consistent with a slow influx of pro-
tons. Subsequent addition of 20
m
Mof either adenosine (Fig. 9B)
or 2-chloroadenosine (not shown) induced a significantly
greater depolarization than controls receiving assay buffer (V
m
5248.8 62.1, p,0.05, 244.1 60.8, p,0.01, and 257.2 6
0.9 mV, respectively).
DISCUSSION
Analysis of the transporter-mediated entry of metabolized per-
meants requires methods that will allow measurements of the
initial rates of influx. Requirements for such methods are de-
finitive 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 mamma-
lian cells (14, 15). Moreover, during this centrifugation period
adenosine influx appears to continue if unlabeled excess per-
meant has not been added. A similar delay of6sincentrifuging
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 (K
m
) for the
transport of adenosine at 22 °C is submicromolar (0.26 60.02
m
M) 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
FIG.5.Effects of ionophores on intracellular pH. BCECF-pre-
loaded cells were suspended in assay buffer, and pH
i
was recorded
fluorimetrically. At indicated times, CCCP (A,10
m
M), nigericin (B,20
m
M), or gramicidin (C,10
m
M) 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 pH
i
level during
the last 45 s of recording. Traces are representative of at least three
similar experiments, and the average effect on pH
i
was used to calcu-
late effects on the protonmotive force.
FIG.6.Effects of ionophores on membrane potential. Procyclic
cells (10
7
) were equilibrated with 0.1
m
Mbis-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
m
MCCCP; B,20
m
Mnigericin; C,
1
m
Mgramicidin; 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 ex-
periments, and the average effect on V
m
, taken as the average potential
during the last minute of recording (gramicidin 30 s), was used to
calculate effects on the protonmotive force.
Adenosine Transport in Trypanosomes 9491

scavenge the purine nucleoside. The specificity of this trans-
porter appears to be solely toward purine nucleosides, with the
naturally occurring nucleosides inosine and guanosine being
potent inhibitors of adenosine influx. Interestingly, the adeno-
sine analogue 8-azidoadenosine showed a large increase (at
least 20-fold) in its apparent K
i
value (5.2 60.07
m
M) 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 per-
meant specificity of the transporter in T. brucei brucei procy-
clics. The fact that [
3
H]inosine was taken up with a K
m
similar
to the K
i
value for inosine on adenosine transport and that
inosine transported was inhibited in turn by adenosine with a
K
i
value identical to the K
m
for adenosine strongly suggests
that inosine and adenosine are taken up by the same trans-
porter. This idea is further supported by the identical actions of
inhibitors ranging from pyrimidine nucleobases to purine nu-
cleoside analogues and by the observation that the slopes of
inhibitor plots was always near 21, 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 adeno-
sine transporter has been suggested to be present in Toxo-
plasma gondii tachyzoites (28), showing a specificity for pu-
rines 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. bru-
cei 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 K
m
and V
max
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 selectiv-
ity is identical to that determined in this study for the adeno-
sine 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 ex-
press at least two purine nucleobase transporters in addition to
the P1 and P2 nucleoside transporters (30). It has been sug-
gested that environmental factors, such as temperature and
pH, are important for transformation into different life cycle
2
H. P. de Koning and S. M. Jarvis, unpublished results.
FIG.7.Correlation between adenosine uptake and protonmo-
tive force. The effects of ionophores and variations of pH
o
on adenosine
uptake (Table II) was plotted against their effects on the PMF. The line
was calculated by linear regression (r
2
50.93); error bars are S.E.
FIG.8.Adenosine enhances the recovery of pH
i
in base-loaded
procyclic T. brucei brucei.Procyclic cells were base-loaded at the
indicated time by the addition of 30
m
lof2MNH
4
Cl to a cuvette
containing '10
7
BCECF-loaded cells in 3 ml of assay buffer. Once a new
stable level of pH
i
was established, adenosine (final concentration 25
m
M,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
pH
i
after adenosine treatment (n58) were taken as the average pH
during the last 60 s of recording and were significantly different from
control (n57) by unpaired Student ttest (7.61 60.048 and 7.77 6
0.056%, respectively, p,0.01).
Adenosine Transport in Trypanosomes9492

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 demon-
strated that adenosine influx in T. brucei brucei procyclics is an
energy-dependent process as exemplified by the inhibitory ef-
fects of the ionophores gramicidin, CCCP, and nigericin. The
energy source was not the transmembrane sodium gradient as
neither monensin, a Na
1
ionophore, nor replacement of Na
1
with NMG
1
had any effect on adenosine transport rates. Sim-
ilarly, 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 electrochem-
ical gradient across the cell membrane, either through cytosolic
acidification (nigericin), membrane depolarization (gramicid-
in), or both (CCCP), inhibited adenosine transport. This finding
indicates that adenosine transport is coupled to the protonmo-
tive force, which consists of both DpH and V
m
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
1
symporter.
Evidence to support this mechanism comes from both direct
and indirect approaches and includes the following. (i) Chang-
ing the external pH from 7.3 to 8.0 inhibited adenosine trans-
port. 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 pH
o
56.4, the pH gradient
across the procyclic membrane is near zero, and the protonmo-
tive force is similar to that observed at pH
o
57.3. (ii) The influx
of adenosine should be accompanied by an electrogenic proton
influx if the carrier is functioning as an adenosine/proton sym-
porter. Direct measurements of pH
i
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
1
-ATPase. However, base loading
with NH
4
Cl 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
1
-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 de-
polarization 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 mamma-
lian 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 fur-
ther 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.
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