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Vol. 59, Issue 3, 586-592, March 2001
Institute of Biomedical and Life Sciences, Division of Infection and Immunity, University of Glasgow, Glasgow, United Kingdom
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results and Discussion
References
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The trypanocidal action of pentamidine is dependent on the rapid, selective accumulation of this drug by the parasite. We have investigated pentamidine transport by the bloodstream and procyclic life cycle stages of Trypanosoma brucei brucei. In bloodstream forms, 50 to 70% of [3H]pentamidine was transported by an adenosine-sensitive pentamidine transporter (ASPT1) that displayed a Km value of 0.26 ± 0.03 µM and Ki values of 0.45 ± 0.04 and 2.5 ± 0.8 µM for adenine and berenil, respectively. These values are very similar to those for inhibition of [3H]adenosine uptake by the P2 adenosine/adenine transporter, suggesting that ASPT1 and P2 may be identical. The remaining 30 to 50% of [3H]pentamidine transport was mediated by a low-capacity high-affinity pentamidine transporter (HAPT1) and a high-capacity low-affinity pentamidine transporter (LAPT1), with Km values of 36 ± 6 nM and 56 ± 8 µM, respectively. HAPT1 was inhibited by propamidine but displayed only low affinity to berenil and stilbamidine, whereas LAPT1 was not inhibited by any of these diamidines. Neither transporter was inhibited by melarsen oxide. In procyclics, an HAPT1-analog (procyclic pentamidine transporter; PPT1) was characterized, but no adenosine-sensitive pentamidine transport could be detected. Treatment with ionophores revealed that PPT1 may be a proton/pentamidine cotransporter.
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Introduction |
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Top
Abstract
Introduction
Experimental Procedures
Results and Discussion
References
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Diamidines, including
pentamidine and berenil (diminazene aceturate), have been first-line
drugs for the treatment of early stage African trypanosomiasis for
decades (Pépin and Milord, 1994
). While their mechanism of action
remains a matter of debate, it is generally accepted that their
selective toxicity for the parasite is the result of accumulation by
the trypanosome, but not the host cell, to very high intracellular
levels (Damper and Patton, 1976a; Carter et al., 1999). Damper and
Patton (1976a,b) first reported that the uptake of pentamidine by
Trypanosoma brucei brucei bloodstream forms is mediated by
high-affinity transporters that are energy-dependent and competitively
inhibited by other diamidines. Carter et al. (1995) tentatively
identified the pentamidine carrier as the P2 adenosine/adenine
transporter that has also been linked to the uptake of the
melaminophenyl arsenical class of trypanocides (Carter and Fairlamb,
1993; Mäser et al., 1999; De Koning et al., 2000a). The
involvement of the P2 transporter in diamidine uptake was also inferred
from the observation that a berenil-resistant clone of T. equiperdum expressed a P2 transporter with much reduced affinity
(Barrett et al., 1995). A biochemical basis for the high-affinity
transport of such diverse molecules as adenosine, pentamidine, and
melarsoprol by the same transporter was provided by the identification
of the substrate recognition motif for P2 (Barrett and Fairlamb, 1999;
De Koning and Jarvis, 1999), which is present on both classes of drugs.
However, significant problems with the hypothesis of pentamidine uptake
by the P2 transporter remained. A 10-fold excess of adenosine failed to
inhibit [3H]pentamidine uptake by T. brucei brucei (Carter et al., 1995), and a cloned T. brucei
brucei transporter with P2-like activity, TbAT1, conferred
sensitivity to arsenicals when expressed in Saccharomyces cerevisiae, but it was not inhibited by pentamidine (Mäser
et al., 1999). In addition, the T. brucei brucei clone RU15,
which did not express measurable P2 transport activity (Carter and
Fairlamb, 1993), was resistant to melaminophenyl arsenicals and
berenil, but not to pentamidine (Fairlamb et al., 1992). Indeed, it
appears that clinical and veterinary isolates as well as laboratory
strains refractory to melaminophenyl arsenicals are usually highly
cross-resistant to berenil but less so to pentamidine (Bacchi, 1993).
Treatment with pentamidine of early stage African sleeping sickness is
not endangered by resistance, although resistance to melarsoprol is a
rapidly growing problem (Legros et al., 1999; Kaminsky and Mäser, 2000). These observations led to the hypothesis that, while berenil and
melaminophenyl arsenicals share a common transporter, pentamidine is
either taken up by a different transporter or by several different transporters, possibly including the P2. In the current study, this
hypothesis was tested by a thorough analysis of
[3H]pentamidine uptake by T. brucei
brucei.
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results and Discussion
References
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Materials. [3H]Pentamidine isethionate was synthesized by Amersham Pharmacia Biotech (3.63 TBq/mmol) for Dr. M. P. Barrett (University of Glasgow) and generously donated for this study. [2,8,5'-3H]Adenosine was from NEN Life Science Products (2.0 TBq/mmol). Most test compounds were purchased from Sigma (St. Louis, MO), including CCCP, nigericin, berenil, and pentamidine (also obtained from Rhône-Poulenc Rorer, Montrouge, France). Melarsen oxide, stilbamidine, and propamidine were generous gifts from Rhône-Poulenc.
Trypanosomes.
T. brucei brucei strain 427 bloodstream trypanosomes were taken from frozen stocks and injected in
adult female Wistar rats. Blood was collected at peak parasitemia by
cardiac puncture under anesthesia. Parasites were isolated from the
blood on a DE52 (Whatman, Maidstone, UK) column (Lanham, 1968)
and washed twice with the assay buffer (33 mM Hepes, 98 mM NaCl, 4.6 mM
KCl, 0.55 mM CaCl2, 0.07 mM
MgSO4, 5.8 mM
NaH2PO4, 0.3 mM
MgCl2, 23 mM NaHCO3, and 14 mM glucose, pH 7.3) by centrifugation at 2000 rpm. Cells were counted
using a hemocytometer and resuspended in assay buffer at
108 cells/ml. Procyclic T. brucei
brucei were cultured in SDM79 culture media as described
(De Koning et al., 1998) and washed into assay buffer as described
above. At the end of each experiment, cell motility was checked under a
phase-contrast microscope.
Transport Studies.
Transport of
[3H]pentamidine and
[3H]adenosine was performed as previously
described with minor modifications (De Koning and Jarvis, 1997a,b,
1999; De Koning et al., 1998). Briefly, 100 µl of assay buffer
containing radiolabel and, where appropriate, test compound in assay
buffer was layered over 250 µl of 7:1 mixture of
di-n-butylphthalate (BDH, Poole, Dorset, UK) and light
mineral oil (Sigma) in a microfuge tube and mixed with 100 µl of
trypanosomes (107 cells, in assay buffer).
Incubations (between 10 and 60 s) were performed at 22 or 0°C
and stopped by the addition of 1 ml of ice-cold 1 mM unlabeled permeant
in assay buffer and centrifugation through the oil (13,000 rpm, 30 s). Tubes were then flash frozen in liquid nitrogen and the bottom
(containing the cell pellet) cut off with a tube cutter. This was
collected in a scintillation tube, incubated for 30 min with 250 µl
of 2% SDS, mixed with 3 ml of Ecoscint A (National Diagnostics,
Atlanta, GA), and shaken well. After incubation overnight at
room temperature, radioactivity was determined by scintillation
counting. Experiments under sodium-free conditions were performed as
described in sodium-free media containing N-methyl-D-glucamine (De Koning et
al., 1998). When permeabilized cells were used, trypanosomes were
placed on ice for 5 min at 5 × 108 cells
ml
1 and incubated with 500 µg
ml1 of lysolecithin for 1 min before 10-fold
dilution with assay buffer. Cells were then washed and resuspended in
fresh assay buffer at 108 cells
ml1 and used for experiment as usual.
equation: Ki = IC50/[1 + (L/Km)], where L is
the permeant concentration and IC50 values were
determined from full dose-response curves with a minimum of eight
points spread over the relevant range. Hill coefficients were always
close to 
1, consistent with monophasic competitive inhibition, except
where indicated. In these cases, data were fitted to equations for
monophasic and biphasic inhibition in the Prism software package and
the two fits compared with an F-test. Data were considered to fit a
two-phase inhibition better when P < 0.05.
In all cases, transport of permeant is understood to mean mediated
transport, after subtraction of radiolabel entering the cell by
diffusion. Diffusion was taken to be the difference in uptake between
an incubation of cells in the presence of 1 mM unlabeled permeant at
room temperature for the same length of time as the rest of the assay,
and a similar incubation with both the cells and permeant kept on ice,
stopped with stop solution immediately after mixing, and spun through
oil. Rates of uptake were calculated from V = (Vmax × L)/(L + Km).
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Results and Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results and Discussion
References
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Characteristics of [3H]Pentamidine Uptake in
Bloodstream Forms of T. brucei brucei.
Uptake of 25 nM [3H]pentamidine in bloodstream forms of
T. brucei brucei was rapid, and linear for at least 3 min,
with a rate of 0.0045 ± 0.0004 pmol (107
cells)1 s1 (Fig.
1A). In the presence of 250 µM
unlabeled pentamidine, the rate was reduced by 97%, indicating that
the vast majority of pentamidine uptake is through a saturable,
carrier-mediated process, with only a very minor diffusion component.
Adenosine (250 µM) inhibited 25 nM
[3H]pentamidine uptake by 65% (Fig. 1A).
Closer examination revealed that adenosine consistently and dose
dependently inhibits 50 to 70% of mediated pentamidine transport, with
a Ki value of 0.80 ± 0.12 µM
(n = 14). Figure 1B shows a typical experiment in which adenosine inhibited ~50% of mediated
[3H]pentamidine (25 nM) transport with a
Ki value of 1.1 µM. In the same
experiment, adenine inhibited pentamidine transport to a similar
degree, with a Ki value of 0.42 ± 0.04 µM (n = 5) (Fig. 1B). The
Ki values of adenosine and adenine on
[3H]pentamidine transport are very similar to
those for adenosine and adenine on
[3H]adenosine transport mediated by the P2
transporter (0.91 ± 0.29 and 0.30 ± 0.02 µM,
respectively) (Carter and Fairlamb, 1993; De Koning and Jarvis, 1999;
De Koning et al., 2000a). It thus appears that pentamidine transport in
T. brucei brucei bloodstream forms is mediated by an
adenosine-sensitive pentamidine transporter (ASPT1), which may be
identical to P2, as well as by one or more adenosine-insensitive
transporters.
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1 (n = 3) and was entirely consistent with a one-transporter (ASPT1) model (Fig. 2).
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); 0.43 ± 0.02 µM (De Koning and Jarvis, 1999)]. While HAPT1 displayed a very high affinity for pentamidine, it also has a
very low capacity, and the order for both
Km and Vmax
values is HAPT1 < ASPT1 < LAPT1.
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;
Waalkes et al., 1970). Over this range, the contribution of LAPT1
increased from 13 to 35% of total pentamidine uptake due to its very
high capacity.
Attempts were made to identify the physiological substrate of the HAPT1
transporter. Up to 1 mM hypoxanthine did not inhibit uptake of 25 nM
[3H]pentamidine in the presence of 250 µM
adenosine (Fig. 3). Other potential
substrates that similarly failed to inhibit HAPT1-mediated pentamidine
transport included inosine, adenine, uracil, uridine, sodium pyruvate
(all at 2.5 mM), the amino acids arginine, lysine, glutamine,
asparagine, phenylalanine, tryptophan (all at 1 mM), and tyrosine (50 µM), the dipeptide Arg-Lys, the polyamines putrescine, spermidine,
and spermine, biotin, folic acid, para-aminobenzoic acid,
and thiamine (all at 1 mM). Each of these compounds was tested in at
least three independent experiments, in triplicate, at a
[3H]pentamidine concentration of 25 nM, and
none produced a statistically significant difference (P < 0.05 in an unpaired Student's t test) from controls with
no inhibitor.
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,b,
1998) are involved in this process, even though pentamidine does
inhibit hypoxanthine uptake in T. brucei brucei bloodstream
forms, with a Ki value of 25 ± 7 µM (De Koning and Jarvis, 1999). High-affinity pentamidine uptake was also
not mediated by polyamine transporters, as has been reported for
Leishmania spp. (Basselin et al., 1997). Inhibition of amino acid transport by pentamidine has been reported in Crithidia
fasciculata (Gutteridge, 1969) and Leishmania spp.
(Basselin et al., 1997), although the latter effect was found to be
noncompetitive. Similar experiments to those described here, but using
[125I]iodopentamidine as permeant, were
performed for LAPT1-mediated pentamidine uptake (De Koning and Jarvis,
unpublished observations).
Affinity of the Pentamidine Transporters for Other Trypanocides. The affinity of the various pentamidine transporters for selected other trypanocides was investigated (Table 1). HAPT1 was inhibited by propamidine but displayed little (berenil and stilbamidine) or no (melarsen oxide) affinity for other antitrypanosome drugs (Fig. 3), and it is not expected to play a role in the uptake of these drugs at therapeutic levels. Similarly, LAPT1 was not inhibited by any of these drugs, unless very high concentrations were used (Table 1). ASPT1, in contrast, was strongly inhibited by melarsen oxide (Fig. 3), berenil, stilbamidine, and propamidine. As shown above for adenine, adenosine, and pentamidine, the ASPT1 Ki values for berenil and melarsen oxide were almost identical to those shown for the P2 transporter (Table 1).
Taken together, all available data are consistent with ASPT1 being identical to P2. The amount of [3H]pentamidine transport inhibited by adenosine varied between experiments. Within a single experiment, however, uptake was inhibited to a very similar degree by diverse P2 substrates, such as adenosine, adenine, melarsen oxide, and various diamidines. As ASPT1 was not inhibited by inosine, the adenosine-sensitive pentamidine uptake was not mediated by the P1 transporter, the only other adenosine carrier in bloodstream trypanosomes (Carter and Fairlamb, 1993; De Koning and Jarvis, 1999).
Thus, the substrate recognition profile of ASPT1 is qualitatively and
quantitatively indistinguishable from P2.
Pentamidine Uptake in Procyclic T. brucei
brucei.
In view of the above results, as well as of
previous studies with diamidine-resistant trypanosomes (see
Introduction), it was deemed very likely that ASPT1 is
identical to the P2 aminopurine transporter. One prediction from this
hypothesis was that ASPT1 would not be present in procyclic forms of
T. brucei brucei, since the P2 transporter is not expressed
in this life cycle stage (De Koning et al., 1998, 2000b). This
prediction was borne out with the observation that transport of 25 nM,
250 nM, or 1 µM [3H]pentamidine by procyclic
T. brucei brucei was not inhibited by up to 1 mM adenosine
or adenine (not shown). Transport was saturable and was linear for up
to 45 min at 1 µM [3H]pentamidine with a rate
of 4.4 pmol (107 cells)1,
whereas at 50 nM uptake proceeded only slightly slower [3.2 pmol
(107 cells)1] and was
linear until all radiolabel was taken up (Fig.
4A). This result indicates that, in
T. brucei brucei procyclics and at 50 nM
[3H]pentamidine, uptake was already proceeding
at near Vmax, consistent with a single
high-affinity transport system rather than the more complex situation
in bloodstream forms.
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1 s1
(n = 8). The very high affinity of this PPT1 is not
statistically different from the Km value
of HAPT1. Moreover, the affinity of the PPT1 and HAPT1 transporters for
other diamidines was also very similar (Table 1). Dixon plots showed
the inhibition of PPT1 by propamidine (Fig. 4B) and by berenil (not
shown) to be competitive. The main difference between the high-affinity
transporters in procyclic and bloodstream T. brucei brucei
was the Vmax value, with the procyclic
trypanosomes displaying a much higher rate of uptake, probably as a
result of a higher level of transporter expression in this life cycle
stage. A low-affinity pentamidine transporter (PPT2) also appeared to
be present in procyclics, but no attempt at further characterization of
this carrier was made, and at <100 nM
[3H]pentamidine concentrations this transporter
did not contribute significantly to the pentamidine uptake. As with
HAPT1-mediated pentamidine uptake, a range of inhibitors was tested in
the hope of identifying the physiological substrate of PPT1. Inosine,
putrescine, spermine, spermidine, biotin, folic acid,
para-aminobenzoic acid, and thiamine were all ineffective at
1 mM. Uracil, lysine, arginine, proline (all at 10 mM), sphingosine
(250 µM), pyruvate (2.5 mM), and 250 µM concentrations of the
peptides Arg-Lys, Arg-Phe, Lys-Lys-Lys, Arg-Ser-Arg, and
(Lys)4 similarly failed to inhibit uptake of 12.5 nM [3H]pentamidine in T. brucei
brucei procyclics (n = 3). While it is impossible
to conclude whether PPT1 and HAPT1 are identical without cloning both
transporters, they are certainly very similar and may represent a
single gene product expressed in both life cycle stages. The mechanism
of high-affinity pentamidine transport was therefore further
investigated in procyclic trypanosomes.
PPT1 is a Proton Symporter.
Although all the data on
[3H]pentamidine uptake in procyclics, and
particularly the linear uptake over short and long intervals, is
consistent with a one-transporter model for high-affinity pentamidine uptake, tests were devised to exclude the possibility of an uptake process driven by binding to an intracellular target. Uptake of 12.5 nM
[3H]pentamidine was determined at 22 and 0°C.
Uptake at 22°C was linear for 100 s at a rate of 0.016 ± 0.003 pmol s1, after which all label had been
taken up. At 0°C, the rate was reduced to 1.6% of the rate at room
temperature (2.5 × 104 ± 2.2 × 105 pmol s1; eight time
points spread over 10 min, r2 > 0.95). The extent of the temperature sensitivity was indicative of a
transporter-mediated mechanism of uptake rather than diffusion.
1 of lysolecithin.
Km and Vmax
values in permeabilized and control cells were determined as described
above. If these kinetic parameters represented binding constants and
maximum rate of binding (limited by diffusion into the cell),
Km would be predicted to be unaltered and
Vmax increased in the permeabilized cells.
However, it was observed that, although the
Km value was very similar to that of
control cells (25 ± 5 versus 40 ± 1 nM, respectively), the Vmax value was reduced by 67% in the
lysolecithin-treated group. In the same experiment,
[3H]adenosine transport was found to be reduced
by 73% after lysolecithin treatment. These results indicate that, like
[3H]adenosine, uptake of
[3H]pentamidine is concentrative, active
transport. The reduced uptake rates following partial permeabilization
may be the result of pentamidine efflux or of the collapse of plasma
membrane ion gradients.
Many transport processes in protozoa (Zilberstein, 1993), including
adenosine and hypoxanthine transport in procyclic T. brucei brucei, are examples of secondary active transport (De Koning and
Jarvis, 1997a; De Koning et al., 1998), with the protonmotive force
supplying the energy for concentrative uptake of nutrients. We
therefore investigated whether the pentamidine transporters described
here are likewise proton symporters. The uptake of pentamidine by PPT1
was independent of sodium, as replacement of Na+
by N-methyl-D-glucamine in the
transport buffer did not affect the rate of 100 nM
[3H]pentamidine uptake over a 5-min period
(0.068 ± 0.005 and 0.072 ± 0.002 pmol
(107 cells)1
s1, respectively; linear regression of eight
points per line, r2 = 0.96 and 0.99).
However, PPT1-mediated pentamidine uptake was inhibited by the proton
ionophore CCCP, with an IC50 value of 4.2 ± 0.3 µM (based on six points up to 40 µM). This value is very
similar to values obtained for inhibition of the U1 and P1 adenosine
transporters in T. brucei brucei uracil procyclics (3.8 ± 0.3 and 2.5 ± 0.4 µM, respectively), which are most probably proton symporters (De Koning and Jarvis, 1998; De Koning et al., 1998).
[3H]Pentamidine transport was also inhibited by
the Na+/H+ exchanger
nigericin and the Na+/K+
exchanger gramicidin (Table 2),
ionophores that acidify the T. brucei brucei cytoplasm or
depolarize the plasma membrane, respectively (De Koning et al., 1998).
These results indicate that reduction of either intracellular pH or
plasma membrane potential will inhibit
[3H]pentamidine uptake, consistent with
PPT1-mediated transport being dependent on the protonmotive force. In
support of this model, NEM and DCCD, sulfhydryl and carboxyl reactive
agents, respectively, which have been shown to inhibit the T. brucei brucei plasma membrane proton pump (Thissen and Wang, 1991;
Defrise-Quertain et al., 1996), also inhibited PPT1-mediated
pentamidine uptake (Table 2). Plotting the effects of NEM, DCCD, and
the ionophores on pentamidine uptake and on protonmotive force against
each other produced a linear correlation (Fig.
5). Pentamidine uptake by HAPT1 in
bloodstream forms was similarly found to be inhibited by CCCP, with 10 µM inhibiting >90% of 12.5 nM
[3H]pentamidine uptake in the presence of 250 µM adenosine.
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Implications for Drug Resistance in African Trypanosomes.
The
presence of two pentamidine transporters in addition to P2 provides an
explanation for why the arsenical-resistant T. brucei brucei
clone RU15, developed from the same strain as used in the present
study, but lacking detectable P2 transporter activity, was only very
slightly cross-resistant to pentamidine (Fairlamb et al., 1992). The
same clone (33-fold-resistant to melarsen oxide in vivo) was
cross-resistant to stilbamidine and berenil (resistance factors of 38- and 32-fold, respectively), which are not transported by HAPT1 and
LAPT1, and only moderately (6-fold) cross-resistant to propamidine,
which may be taken up by HAPT1. It thus appears that the model of three
distinct diamidine transporters presented here could form a basis to
explain cross resistance patterns of drug resistance, at least in
T. brucei brucei, and possibly other African trypanosomes.
In agreement with such a model, clinical resistance to melarsoprol is a
fast-growing problem, while resistance to pentamidine or suramin is
reportedly all but absent (Kaminsky and Mäser, 2000). In
laboratory strains with induced arsenical resistance, cross-resistance
to berenil is also more widespread than to pentamidine (Bacchi, 1993).
However, while this model may be relevant in many cases of drug
resistance in African trypanosomes, it must be noted that not all
observed drug resistance is necessarily due to changes in transporter
function. The T. brucei brucei strain PR32.6 described by
Berger et al. (1995) was resistant to pentamidine but not deficient in
pentamidine accumulation or P2 transporter activity. It should also be
noted that melarsoprol-pentamidine cross-resistance does occur in some
laboratory strains (Kaminsky and Mäser, 2000). In most cases,
however, the resistance had been induced by prolonged exposure to
sublethal doses of melarsoprol, a treatment that is highly mutagenic,
and may induce changes in additional transporters along with P2. A well
documented example is the 6-fold reduction in P1-mediated adenosine
transport in RU15 (Carter and Fairlamb, 1993; De Koning et al., 2000a).
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Acknowledgments |
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I thank Prof. Keith Gull (University of Manchester, UK) for the use of research facilities. I am indebted to Lynsey McMurray for expert technical assistance, to Dr. M.P. Barrett (University of Glasgow, UK) for the kind gift of [3H]pentamidine, and to Drs. Simon Jarvis (University of Kent, UK), Pat Bray (University of Liverpool, UK), and Keith Matthews (University of Manchester, UK) for helpful discussions.
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Footnotes |
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Received September 13, 2000; Accepted December 1, 2000
Most of this work was funded by the Wellcome Trust, but part was performed at the School of Biological Sciences of the University of Manchester with the financial support of the Research Division of Biochemistry.
Send reprint requests to: Harry P. De Koning, Inst. of Biomedical and Life Sciences, Division of Infection and Immunity, Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, UK. E-mail: h.de-koning{at}bio.gla.ac.uk
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Abbreviations |
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ASPT1, adenosine-sensitive pentamidine transporter-1; CCCP, carbonyl cyanide chlorophenylhydrazone; HAPT1, high-affinity pentamidine transporter-1; LAPT1, low-affinity pentamidine transporter-1; PPT, procyclic pentamidine transporter; DCCD, N,N'-dicyclohexylcarbodiimide; NEM, N-ethylmaleimide.
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References |
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Top
Abstract
Introduction
Experimental Procedures
Results and Discussion
References
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H. P. de Koning, L. F. Anderson, M. Stewart, R. J. S. Burchmore, L. J. M. Wallace, and M. P. Barrett The Trypanocide Diminazene Aceturate Is Accumulated Predominantly through the TbAT1 Purine Transporter: Additional Insights on Diamidine Resistance in African Trypanosomes Antimicrob. Agents Chemother., May 1, 2004; 48(5): 1515 - 1519. [Abstract] [Full Text] [PDF]
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M. L. Stewart, G. J. Bueno, A. Baliani, B. Klenke, R. Brun, J. M. Brock, I. H. Gilbert, and M. P. Barrett Trypanocidal Activity of Melamine-Based Nitroheterocycles Antimicrob. Agents Chemother., May 1, 2004; 48(5): 1733 - 1738. [Abstract] [Full Text] [PDF]
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 B. Nguyen, D. Hamelberg, C. Bailly, P. Colson, J. Stanek, R. Brun, S. Neidle, and W. D. Wilson Characterization of a Novel DNA Minor-Groove Complex Biophys. J., February 1, 2004; 86(2): 1028 - 1041. [Abstract] [Full Text] [PDF]
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 E. Matovu, M. L. Stewart, F. Geiser, R. Brun, P. Maser, L. J. M. Wallace, R. J. Burchmore, J. C. K. Enyaru, M. P. Barrett, R. Kaminsky, et al. Mechanisms of Arsenical and Diamidine Uptake and Resistance in Trypanosoma brucei Eukaryot. Cell, October 1, 2003; 2(5): 1003 - 1008. [Abstract] [Full Text] [PDF]
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M. I. Al-Salabi, L. J. M. Wallace, and H. P. de Koning A Leishmania major Nucleobase Transporter Responsible for Allopurinol Uptake Is a Functional Homolog of the Trypanosoma brucei H2 Transporter. Mol. Pharmacol., April 1, 2003; 63(4): 814 - 820. [Abstract] [Full Text] [PDF]
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 A. Lansiaux, F. Tanious, Z. Mishal, L. Dassonneville, A. Kumar, C. E. Stephens, Q. Hu, W. D. Wilson, D. W. Boykin, and C. Bailly Distribution of Furamidine Analogues in Tumor Cells: Targeting of the Nucleus or Mitochondria Depending on the Amidine Substitution Cancer Res., December 15, 2002; 62(24): 7219 - 7229. [Abstract] [Full Text] [PDF]
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M. Basselin, H. Denise, G. H. Coombs, and M. P. Barrett Resistance to Pentamidine in Leishmania mexicana Involves Exclusion of the Drug from the Mitochondrion Antimicrob. Agents Chemother., December 1, 2002; 46(12): 3731 - 3738. [Abstract] [Full Text] [PDF]
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L. J. M. Wallace, D. Candlish, and H. P. De Koning Different Substrate Recognition Motifs of Human and Trypanosome Nucleobase Transporters. SELECTIVE UPTAKE OF PURINE ANTIMETABOLITES J. Biol. Chem., July 12, 2002; 277(29): 26149 - 26156. [Abstract] [Full Text] [PDF]
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) or 250 µM unlabeled pentamidine (
). Control cells were
incubated with the same volume of assay buffer without inhibitor (
).
B, inhibition of 25 nM [3H]pentamidine by various
concentrations of adenosine (
, solid
line), and pentamidine (
),
propamidine (
,
IC50 = 110 µM), and berenil (
,
IC50 = 78 µM).
