* Zentrum für Infektionsforschung, Institut für Physiologie der Universität Würzburg, D-97070 Würzburg, Germany
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Abstract |
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Here we describe the identification and characterization of a physiological marker that is associated with the chloroquine-resistant (CQR) phenotype in the human malarial parasite Plasmodium falciparum. Single cell in vivo pH measurements revealed that CQR parasites consistently have an elevated cytoplasmic pH compared to that of chloroquine-sensitive (CQS) parasites because of a constitutively activated Na+/H+ exchanger (NHE). Together, biochemical and physiological data suggest that chloroquine activates the plasmodial NHE of CQS parasites, resulting in a transitory phase of rapid sodium/hydrogen ion exchange during which chloroquine is taken up by this protein. The constitutively stimulated NHE of CQR parasites are capable of little or no further activation by chloroquine. We propose that the inability of chloroquine to stimulate its own uptake through the constitutively activated NHE of resistant parasites constitutes a minimal and necessary event in the generation of the chloroquine-resistant phenotype.
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Introduction |
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CHLOROQUINE was the first choice antimalarial drug for more than three decades until the emergence and spread of chloroquine-resistant Plasmodium falciparum strains rendered its application ineffective in many parts of the world. As other available antimalarial drugs are not as effective, safe, or affordable as chloroquine, the incidence of malaria has soared to ~500 million clinical cases per year (World Health Organization, 1996). In understanding the molecular mechanism of chloroquine resistance, we may gain valuable insights into the parasite's biology, which, in turn, may inspire rational programs for the development of novel antimalarial drugs with improved pharmacological properties.
Chloroquine targets the intraerythrocytic stages of P. falciparum (Yayon et al., 1983), which feed on the erythrocyte's hemoglobin. The toxic heme moiety released in the
process is polymerized in the parasite's acidic vacuole into
insoluble and inert hemozoin (Slater et al., 1991
). Chloroquine, accumulating to high concentrations in the vacuole,
exerts its specific antimalarial effect in the inhibition of
heme polymerization (Slater and Cerami, 1992
; Dorn et al.,
1995
; Sullivan et al., 1996
). Chloroquine-resistant (CQR)1
parasites accumulate less chloroquine in their vacuoles
than do chloroquine-sensitive (CQS) parasites (Fitch, 1970
,
1973
), suggesting that a reduction in the vacuolar chloroquine concentration, below levels necessary to inhibit heme
polymerization, is responsible for chloroquine resistance.
Two different models have been proposed to explain the
differences in chloroquine accumulation associated with
the resistant phenotype. The first model invokes the acquisition of a rapid chloroquine efflux mechanism by CQR
parasite isolates (Krogstad et al., 1987; Martin et al., 1987
).
The second model proposes that CQR parasites have an
elevated pH in their acidic lysosomes that would reduce
acidotropic accumulation of the diprotic weak base chloroquine (Ginsburg and Stein, 1991
).
We have recently presented compelling evidence in favor
of a third model (Sanchez et al., 1997). We found that a
carrier-mediated import mechanism is responsible for chloroquine uptake and accumulation in P. falciparum, in contrast to uninfected erythrocytes where chloroquine is solely
taken up by nonionic diffusion of the free base (Ferrari
and Cutler, 1990
; Sanchez et al., 1997
). The observation that carrier-mediated chloroquine uptake is competitively
inhibited by 5-(N-ethyl-N-isopropyl) amiloride (EIPA), a
specific and reversible inhibitor of eukaryotic Na+/H+ exchangers (Vigne et al., 1983
; Kleyman and Cragoe, 1990
),
identified the plasmodial Na+/H+ exchanger (NHE) as a
primary candidate for the chloroquine importer (Sanchez
et al., 1997
). The P. falciparum NHE resides in the parasite's plasma membrane, where it plays an essential role in
the maintenance of the parasite's cytoplasmic pH, expelling excess protons generated during metabolism in exchange for sodium ions (Bosia et al., 1993
).
The genetic linkage between the CQR phenotype and
a reduction in carrier-mediated chloroquine uptake suggests that the P. falciparum NHE is altered in CQR parasites (Sanchez et al., 1997). To verify this hypothesis, we
have studied the pH-regulating function of the NHE as
well as its putative role in chloroquine transport, in both
CQS and CQR parasites. We found that a change in NHE
activity, resulting in an elevated cytoplasmic pH, is genetically linked with the CQR phenotype. We further provide
evidence for the model that the activity status of the NHE
determines the ability of this protein to import chloroquine.
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Materials and Methods |
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P. falciparum Culture
The P. falciparum isolates investigated were cultured in vitro as described
(Trager and Jensen, 1976) and then synchronized using the sorbitol
method (Lambros and Vanderberg, 1979
).
Fluorimetric Assay of Intracellular pH
Fluorimetric in vivo cytoplasmic pH measurements were performed using
the fluorochrome 2,7
-bis-(2-carboxyethyl)-5,6-carboxyfluorescein-acetoxymethylester (BCECF-AM; Molecular Probes, Inc., Eugene, OR) as
described (Weiner and Hamm, 1989
; Wünsch et al., 1995
). Briefly, intraerythrocytic P. falciparum cultures were collected and washed twice in
Ringer solution (122.5 mM NaCl, 5.4 mM KCl, 1.2 mM CaCl2, 0.8 mM
MgCl2, 5.5 mM D-glucose, 1.0 mM Na2HPO4, 10 mM Hepes, pH 7.4, at
37°C). The cells were incubated for 3 min in Ringer solution containing 3 µM of BCECF-AM. The erythrocytes were seeded onto poly-L-lysine (Sigma Chemical Co.)-coated coverslips and then rinsed with Ringer solution to remove excess BCECF-AM. The coverslip was mounted in a superfusion chamber on the stage of an inverted microscope (model 100 TV
Axiovert; Carl Zeiss, Inc., Thornwood, NY), and continuously superfused
with Ringer solution prewarmed to 37°C to maintain physiological conditions. Fluorescence emissions at 520 nm were monitored from alternate
excitation at wavelengths of 488 and 460 nm, using an automatic filter
changing device (Carl Zeiss, Inc.). Using the ratio of the fluorescence signals at the two excitation wavelengths, a pH reading was obtained independently of changes in either cell volume or fluorochrome concentration during the measurement (Weiner and Hamm, 1989
; Negulescu et al., 1990). Data acquisition were controlled using Attofluor software (Carl
Zeiss, Inc.). This software allows us to define arbitrary areas of interest
within a cell where the pH is measured. For each area of interest, a separate pH calibration is performed that compensates for quenching effects
and variations in the fluorescence signal intensities emitted from different
areas. Calibration of intracellular pH was performed by the nigericin/high
potassium method using at least two different buffers of known pH (Thomas et al., 1979
). The pH was exclusively determined in the cytoplasm of late
trophozoite stage parasites (24-30 h after invasion) positioned horizontally on the slide, as determined by light microscopy. The area where the
pH is measured was always defined completely within the parasite's cytoplasm. Where indicated, the Ringer solution was replaced by a bicarbonated buffer (106 mM NaCl, 24 mM NaHCO3, 5.4 mM KCl, 1.2 mM CaCl2,
0.8 mM Na2HPO4, 0.2 mM NaH2PO4, 0.8 mM MgCl2, 5.5 mM D-glucose,
pH 7.4, at 37°C, gassed with CO2). Chloroquine exhibits no chromatic activity at the wavelengths used to measure the cytoplasmic pH or sodium
ion concentration (data not shown).
Fluorimetric pH Measurements of Isolated P. falciparum Parasites
P. falciparum-infected erythrocytes were loaded with BCECF as described above. The host erythrocyte was lysed using the dipeptide glycyl-
L-serine at a concentration of 5% (Elford, 1993), and then solubilized in a
modified Hepes Ringer buffer (115 mM NaCl, 10 mM KCl, 1.2 mM CaCl2,
0.8 mM MgCl2, 5.5 mM D-glucose, 1.0 mM Na2HPO4, 10 mM Hepes, pH
7.1, at 37°C). The modified buffer resembles the cytoplasmic ionic environment of the host erythrocyte (Lee et al., 1988
). Isolated parasites were
then seeded on coverslips and analyzed as described above.
Determination of NHE Activity
NHE activity was determined using the ammonium chloride prepulse
technique as described (Boyarsky et al., 1990). Briefly, the parasite's cytoplasmic pH was monitored while the infected erythrocyte was superfused
with Ringer solution containing 40 mM of NH4Cl for 2 min. The superfusion buffer was then changed to Ringer solution alone and then the time
course of the cytoplasmic pH recovery was monitored. To determine the
component of proton flux that is independent of NHE activity, 50 µM of
EIPA was added at different time points during the pH recovery phase as
described previously (Boyarsky et al., 1990
). The EIPA-independent proton flux was subsequently subtracted from the net proton flux. NHE activity was calculated by multiplying the EIPA-sensitive proton flux with the
corresponding pH-dependent intracellular buffer capacity
i. The pH-dependent
i was determined as described (Boyarsky et al., 1988
). Briefly,
cells were superfused with Ringer solution containing 40, 20, 10, 5, and
0 mM of NH4Cl while the cytoplasmic pH of the parasite was monitored.
The
i was calculated for each of the different NH4Cl concentrations by
dividing the change in the intracellular NH4+ concentration ([NH4+]i) by
the corresponding change in the cytoplasmic pH (
pHi). [NH4+]i was calculated using the Henderson-Hasselbalch equation, assuming that free
NH3 is in equilibrium across the plasma membrane.
Fluorimetric Assay of the Intracellular Sodium Ion Concentration
Noninvasive fluorimetric measurements of the cytoplasmic sodium ion
concentration were made using the fluorochrome SBFI (sodium-binding benzofuran isophthalate-acetoxymethylester; Molecular Probes, Inc.) as a
sodium ion indicator as described (Minta and Tsien, 1989; Negulescu and
Machen, 1990
). Briefly, intraerythrocytic P. falciparum cultures were
maintained for 2 to 3 h in medium containing 10 µM SBFI-AM, dissolved
in Pluronic F127 (10% wt/vol DMSO; Molecular Probes, Inc.). Cytoplasmic sodium ion concentrations were then measured using the ratio imaging system described above, with the exception that the fluorescence emissions at 520 nm were monitored at alternate excitation wavelengths of 334 and 380 nm. For internal calibration, cells were permeabilized by the addition of 10 µM of gramicidin followed by superfusion with at least four different buffers of known sodium ion concentration. The buffers for calibration were made by mixing different amounts of the sodium-free buffer A
(130 mM K-gluconate, 30 mM KCl, 1.2 mM CaCl2, 0.8 mM MgCl2, 5.5 mM
D-glucose, 10 mM Hepes, pH 7.1, at 37°C) with the potassium-free buffer
B (130 mM Na-gluconate, 30 mM NaCl, 1.2 mM CaCl2, 0.8 mM MgCl2, 5.5 mM D-glucose, 10 mM Hepes, pH 7.1, at 37°C).
Determination of the IC50 Values for Chloroquine
The susceptibility of the P. falciparum clones investigated to chloroquine
was determined according to the WHO guidelines (Wernsdorfer and
Payn, 1988). Briefly, synchronized cultures containing ring stage parasites
at a parasitemia of 1% were aliquoted into microtiter plates and serial dilutions of chloroquine were added. After incubation for 24 h, the medium
was changed and replaced by medium containing the same chloroquine
concentration supplemented with [3H]hypoxanthine (2 µCi/ml; Amersham Corp., Arlington Heights, IL). Cells were harvested after a further
24 h of incubation. The incorporation of [3H]hypoxanthine into the parasite's DNA, which occurs during the trophozoite and early schizont stages,
was then measured as a function of the chloroquine concentration present
in the medium, and then a 50% inhibitory concentration of chloroquine
(IC50) was determined. The IC50 value is the chloroquine concentration that inhibits 50% of the parasites in their development from rings to schizonts within a 48-h period. A parasite clone is considered chloroquine resistant if the IC50 value exceeds 160 nM (Wernsdorfer and Payn, 1988
).
[3H]Chloroquine Uptake Kinetics
The kinetics of chloroquine uptake in the presence of various NHE inhibitors was determined as previously described (Sanchez et al., 1997). The
NHE inhibitors examined were: amiloride (Sigma Chemical Co.); DMA
(5-[N,N-dimethyl]amiloride; Sigma Chemical Co.); EIPA (5-[N-ethyl-
N-isopropyl]amiloride; Hoechst A.G. Frankfurt, Germany); HMA (5-[N,N-hexamethylene]amiloride; Molecular Probes, Inc.); Hoe 370 (5-chloro-
2-indoloyl guanidine; Hoechst); IBMA (5-[N-isobutyl-N-methyl]amiloride;
Molecular Probes Inc.).
Statistical Analysis
Data were evaluated for statistical significance using the Student's paired t test or the ANOVA test as appropriate. Significance was assumed if P < 0.05. Values are given as the mean of (n) independent measurements ± SEM.
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Results |
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Chloroquine Uptake Is Mediated by an NHE in P. falciparum
Based on our finding that chloroquine uptake by P. falciparum is a carrier-mediated process that is competitively
inhibited by EIPA, we have recently formulated the hypothesis that the P. falciparum NHE mediates chloroquine uptake (Sanchez et al., 1997). To confirm this hypothesis we
determined the effect of other NHE inhibitors on chloroquine uptake. Five NHE inhibitors were chosen: amiloride,
DMA, IBMA, HMA, and Hoe 370. Like EIPA, DMA,
IBMA, and HMA are amiloride derivatives, whereas Hoe
370 belongs to a structurally unrelated group of NHE
inhibitors that are derived from indoloyl-guanidine. The
initial velocities of [3H]chloroquine uptake by the CQS
parasite clone HB3 were monitored over a range of chloroquine concentrations in the presence of each of these
NHE inhibitors. For each NHE inhibitor, three different concentrations were examined and then the data obtained
were analyzed using Lineweaver-Burk plots (Fig. 1). It
was found that all the NHE inhibitors examined inhibit
chloroquine uptake in a strictly competitive manner. This
finding indicates that the chloroquine and NHE inhibitors
examined compete for binding to the same site. For each inhibitor, the apparent constant of inhibition (Ki) was determined by plotting the slopes of the lines versus the corresponding inhibitor concentrations. The apparent Ki values
obtained range from 450 µM for amiloride, to 8 µM for
IBMA, and to 3 µM for HMA (Table I). This establishes a
potency scale with HMA being the most potent inhibitor
of chloroquine uptake and amiloride being the least (Table I). The ability of these substances to inhibit eukaryotic
NHEs is ranked in the same order (Kleyman and Cragoe, 1988
, 1990
), providing compelling evidence in support of
our proposal that the P. falciparum NHE mediates chloroquine uptake.
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Fluorimetric Measurement of Cytoplasmic pH in P. falciparum
As the CQR phenotype is genetically linked with changes
in the chloroquine uptake kinetics (Sanchez et al., 1997),
this finding suggests that the NHE is altered in response to
chloroquine selection. These alterations may also affect
the role of the NHE in pH regulation, implying that cytoplasmic pH regulation may differ between CQS and CQR
parasite clones. Current protocols to measure cytoplasmic pH rely on single-cell noninvasive fluorimetric techniques
that use the pH-sensitive fluorochrome BCECF and a ratio-imaging system to detect changes in the spectral properties of BCECF (Weiner and Hamm, 1989
; Wünsch et al.,
1995
). We initially determined if such a fluorimetric technique can be applied in measuring the cytoplasmic pH (pHi)
of P. falciparum while still residing within its host erythrocyte. After loading with BCECF, a strong and readily detectable fluorescence signal was observed in infected erythrocytes in the area of the parasite, as independently
identified by light microscopy (Fig. 2 A). The intensity of
the fluorescence signal from the parasite was found to be
four times higher per squared micrometer than that emitted by the host erythrocyte cytoplasm (Fig. 2 B). More
than 90% of the signal remained associated with the parasite even after lysis of its host erythrocyte, using the dipeptide glycyl-L-serine (Elford, 1993
; Fig. 2 B). These data indicate that in the area where the parasite lies, most, if not all, of the fluorescence signal is emitted by the parasite itself. The contribution of the host erythrocyte cytoplasm is
<0.03 pH units, as calculated from the thickness of the
erythrocyte cytoplasm above the parasite of ~0-0.125 µm
(Rosenthal et al., 1988
; Sam-Yellowe et al., 1988
), and the
fluorescence signals emitted from the parasite both within
and without the host erythrocyte.
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A pHi of 7.34 ± 0.02 (n = 10) was determined for the
CQR parasite clone FCR3-A2 (Fig. 2 D), which is identical to the pHi value obtained for FCR3-A2 after permeabilization of the host erythrocyte (Bosia et al., 1993). The
pHi simultaneously determined in the cytoplasm of the
host erythrocyte is 7.12 ± 0.02 (n = 5). This pHi value is
slightly higher than that of uninfected erythrocytes (7.05 ± 0.02 (n = 121); Fig. 2, C and D), which is not entirely unexpected since the parasite extensively modifies its host cell including band 3 (Cl
/HCO3
exchanger), which plays a
major role in erythrocyte pH maintenance (Crandall and
Sherman, 1991
). These data indicate that the noninvasive fluorimetric method used in this study allows us to reliably
determine the cytoplasmic pH of single, living P. falciparum parasites still within their host erythrocyte.
An Elevated Cytoplasmic pH Is Genetically Linked with the CQR Phenotype
The cytoplasmic pH of two P. falciparum clones were determined: HB3, a fully chloroquine susceptible clone from
Honduras (IC50 = 81 nM); and Dd2, a CQR clone from Indochina (IC50 = 733 nM). All pHi measurements were
made on late trophozoite-infected erythrocytes. A clear
difference in the pHi was found between these two clones. The CQS clone HB3 has a pHi of 7.18 ± 0.02 (n = 39),
whereas the CQR clone Dd2 has a significantly higher pHi
of 7.32 ± 0.02 (n = 47; P < 0.05; Table II). The differences
in cytoplasmic pH observed between HB3 and Dd2 are independent of the superfusion buffers used during the measurement, as the same phenomenon was observed using a
CO2-pressurized bicarbonated buffer instead of a Hepes buffer (Table II). However, the pHi values obtained are
slightly higher using the CO2-pressurized bicarbonated superfusion buffer, as has also been observed in other systems (Saarikoski et al., 1997; Boyarsky et al., 1988
).
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To exclude the possibility that the differences in the cytoplasmic pH values observed between HB3 and Dd2 result from variations in the ionic or proteinatious environment of their respective host erythrocytes, we isolated
these parasites from their host cells using the dipeptide
glycyl-L-serine (Elford, 1993). pH measurements were
then performed on single isolated parasites as described above. A pHi of 7.22 ± 0.02 was obtained for HB3, and a
pHi of 7.37 ± 0.02 for Dd2 (Table II). These data confirm
the observation that the CQR parasite clone Dd2 has a significantly higher cytoplasmic pH than does the CQS parasite clone HB3. Furthermore, as the cytoplasmic pH values determined for isolated parasites are comparable to those obtained for live parasites still within their host erythrocytes, this experiment further validates the accuracy of
the noninvasive fluorimetric technique used in this study.
To investigate the possibility of a genetic linkage between the CQR phenotype and a change in the cytoplasmic pH, we examined a genetic cross made with Dd2 and
HB3 as parental clones (Wellems et al., 1990, 1991
). From
this cross 16 independent progeny clones were investigated; 8 are CQS, 8 CQR. The cytoplasmic pH was determined for each of the progeny clones and two groups were
clearly identified (Fig. 3 A). All 8 CQS progeny clones
share the same cytoplasmic pH as the CQS parental clone
HB3. Likewise, the cytoplasmic pH of the 8 CQR progeny
clones was the same as that of the CQR parental clone
Dd2. Thus, differences in the cytoplasmic pH are genetically linked with the CQR phenotype in the genetic cross
between HB3 and Dd2.
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Epidemiological studies have suggested that chloroquine resistance emerged simultaneously in two foci
Latin America and Southeast Asia (Payne, 1987
). This
finding begs the question whether CQR parasite clones
from geographic origins other than Southeast Asia also
have an elevated cytoplasmic pH. To address this question we measured the cytoplasmic pH of 17 geographically dispersed CQS and CQR P. falciparum clones (Table III). It
was found that the 11 CQR parasite clones investigated
have a significantly higher cytoplasmic pH than the 6 CQS
parasite clones investigated (P < 0.05), independent of
their geographic origin (Fig. 3 B).
Characteristics of NHE Differ between CQS and CQR Parasites
The elevated cytoplasmic pH of CQR parasites is consistent with our proposal that the NHE has been altered in
response to chloroquine selection. To verify this hypothesis, we investigated the kinetic and physiological properties of the NHEs in both the CQS parasite clone HB3 and
the CQR parasite clone Dd2, using the ammonium chloride prepulse technique (Boyarsky et al., 1990; Fig. 4 A).
Although the infected erythrocyte is a two-compartment
system, no differences in the temporal pH changes were
observed between the host erythrocyte cytoplasm and the
parasite cytoplasm during the ammonium chloride pulse.
This is most likely a consequence of the short distance of
0-0.125 µm between both compartments (Rosenthal et al.,
1988
; Sam-Yellowe et al., 1988
) and the speed of 0.21 cm/s
at which NH3 migrates through the erythrocyte (Labotka et al., 1995
). The contribution of proton fluxes, other than
those resulting from NHE activity, were determined in the
presence of 50 µM of EIPA, which was added during the
pHi recovery phase at various time points (Fig. 4 B). The
identity of the cytoplasmic acid-loading process unmasked
by EIPA is not yet known. It could result from metabolic
generation of protons, influx of protons, and/or chloride-
base exchange.
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The NHE activities of both HB3 and Dd2 were quantified by multiplying the EIPA-sensitive component of the
proton flux for each parasite clone with the appropriate
value of the intracellular buffer capacity of the cytoplasm
i (Boyarsky et al., 1990
).
i was determined for both HB3
and Dd2 using stepped ammonium chloride gradients (Boyarsky et al., 1988
; Fig. 4 C). No differences in
i were observed between the CQS parasite clone HB3 and the CQR parasite clone Dd2 (Fig. 4 D). The activity of the NHE is
given in terms of protons extruded per liter of parasites
per minute at a defined cytoplasmic pH (Fig. 5). A sigmoid
relationship, in both HB3 and Dd2, was found between
the NHE activity and the cytoplasmic pH , as also seen in
other eukaryotes (Noel and Pouyssegur, 1995
; Fig. 5). However, the pH-dependent activity of the NHEs differ between HB3 and Dd2. At any given cytoplasmic pH, the
CQR parasite clone Dd2 has a more active NHE than
does the CQS parasite clone HB3. Furthermore, the NHE
of Dd2 has an extended pH working range as it continues
to be active at higher cytoplasmic pH values than that of
HB3. This finding indicates that the set point, the pH
value at which NHE ceases activity (Kaila and Vaughan-Jones, 1987
), is shifted towards an alkaline pH in Dd2.
Thus, the differences in cytoplasmic pH between CQS and
CQR parasite clones can be attributed to NHEs with different set points (Noel and Pouyssegur, 1995
).
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Stimulation of the NHE by Chloroquine
We next examined what role the altered properties of the NHE play in the ability of this protein to mediate chloroquine uptake. On the addition of 50 nM of chloroquine to the CQS parasite clone HB3, the pHi increased from 7.18 ± 0.02 to a new steady-state cytoplasmic pH of 7.40 ± 0.03 (n = 19, P < 0.05; Fig. 6 A). In comparison, only a small increase in pHi from 7.32 ± 0.02 to 7.38 ± 0.03 (n = 19, P < 0.05) occurred in the CQR parasite clone Dd2 (Fig. 6A). Experiments with other CQS and CQR parasite clones confirmed these data, as all CQS parasite clones investigated respond to the addition of chloroquine with a strong alkalization, whereas all the CQR parasite clones showed only a small increase in cytoplasmic pH (Table III).
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Interestingly, the alkalization induced by chloroquine in the cytoplasm of CQS parasite clones is accompanied by a transient acidification of the host cell cytoplasm (iRBC) from 7.11 ± 0.02 (n = 6) to a peak value of 6.98 ± 0.03 (n = 6, P < 0.05) 5 min after chloroquine addition (Table III). In comparison, no significant change in the cytoplasmic pH of uninfected erythrocytes (RBC) was observed upon the addition of 50 nM of chloroquine (Fig. 6).
The chloroquine-induced cytoplasmic alkalization observed in CQS parasite clones is fully inhibited by EIPA.
A slight acidification is observed in the cytoplasm of HB3
when both chloroquine and EIPA were added simultaneously (Fig. 7 A), a finding consistent with the inhibition
of NHE activity (Noel and Pouyssegur, 1995). Together,
these data suggest that the chloroquine-induced cytoplasmic pH changes, i.e., alkalization in the parasite and acidification of the host erythrocyte, result from the activity of
the parasite's NHE, rather than the consumption of protons by chloroquine-free base, which may have crossed the
parasite membrane by nonionic diffusion.
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As the NHE transports protons in exchange for sodium
ions, changes in NHE activity may not only affect the cytoplasmic pH but also the cytoplasmic sodium ion concentration (Nai), as shown in other systems (Borin and Siffert,
1990; Ye et al., 1996
). To explore this hypothesis, we determined the cytoplasmic sodium ion concentration of HB3
in the presence and absence of chloroquine, using the fluorochrome SBFI as a sodium ion indicator and the ratio imaging system to detect changes in the spectral properties of
SBFI (Minta and Tsien, 1989
; Negulescu and Machen, 1990
).
It was found that upon the addition of 50 nM of chloroquine the cytoplasmic sodium ion concentration increased
in HB3 from 21 ± 1 mM to 65 ± 4 mM (n = 10; Fig. 7 B).
EIPA completely inhibits the chloroquine-induced increase in Nai (Fig. 7 B). In comparison, no change in the
cytoplasmic sodium ion concentration was observed for
the CQR parasite clone Dd2 upon the addition of chloroquine (data not shown).
To establish the temporal relationship between chloroquine uptake and the chloroquine-induced changes in pH and sodium ion concentration, we determined the time course of [3H] chloroquine uptake in both HB3 and Dd2 (Figs. 6 B and 7 C). A comparative analysis clearly reveals a coincidence of these events. As chloroquine enters the parasite, protons are extruded from the parasite's cytoplasm into the host erythrocyte's cytoplasm, while sodium is taken up. As all three events are completely inhibited in the presence of EIPA (Fig. 7), this suggests that the plasmodial NHE mediates chloroquine uptake together with sodium and in exchange for protons.
However, the amount of chloroquine taken up by HB3, ~40 µM, could not readily account for the degree of alkalization and hypernaturesis observed in the cytoplasm of HB3 should we assume a simple exchange reaction. Therefore, we explored the possibility that the NHE is activated by chloroquine, giving rise to the extent of the cytoplasmic alkalization and hypernaturesis observed. To verify this hypothesis, we determined the NHE activities of both HB3 and Dd2 in the presence of 50 nM of chloroquine. It was found that chloroquine activates the NHE of HB3, causing an increase in its pH-dependent activity and pH working range (Fig. 8). Thus, in the presence of chloroquine, the NHE of the CQS parasite clone HB3 acquires kinetic and physiological properties similar to those of the CQR parasite clone Dd2. As a result of this activation, large quantities of protons are exchanged for sodium ions, giving rise to the cytoplasmic alkalization and hypernaturesis observed. Conversely, no significant stimulatory effect was exerted by chloroquine on the NHE of the CQR parasite clone Dd2 (data not shown). These findings establish a link between the extent of chloroquine uptake and the ability of chloroquine to activate the P. falciparum NHE.
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Discussion |
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Chloroquine exerts its specific antimalarial activity after
accumulation within the human malarial parasite P. falciparum. Although chloroquine can permeate membranes
by nonionic diffusion, and subsequently accumulates in
acidic subcellular compartments because of its diprotic
weak base properties (Yayon et al., 1984), the extent of
chloroquine uptake by P. falciparum cannot be adequately explained by a diffusion-controlled process (Ferrari and
Cutler, 1991
). P. falciparum accumulates several orders of
magnitude more chloroquine than any other eukaryotic
cell, including those similar to P. falciparum that contain
large acidic vacuoles (Krogstad et al., 1992
; MacIntyre and
Cutler, 1993
). Furthermore, the kinetics of chloroquine
uptake by P. falciparum is inconsistent with a diffusion-controlled mechanism as it is temperature-sensitive, saturable, and inhibitable (Sanchez et al., 1997
). These findings suggest that a mechanism other than nonionic diffusion,
driven by the acidotropic properties of chloroquine, is responsible for chloroquine uptake and accumulation in P. falciparum. We have recently demonstrated that chloroquine uptake is carrier-mediated (Sanchez et al., 1997
).
Here we present several lines of evidence that suggest the
chloroquine importer is the P. falciparum NHE, a plasma
membrane protein involved in cytoplasmic pH and cell volume regulation.
Firstly, chloroquine uptake is competitively inhibited by
a broad range of NHE inhibitors, including the amiloride
derivatives, DMA, EIPA, IBMA, and HMA, as well as the
structurally unrelated indoloyl guanidine derivative Hoe
370 (Fig. 1). The apparent constants of inhibition observed
vary amongst the NHE inhibitors examined, defining a potency scale with HMA being the most potent inhibitor of
chloroquine uptake and amiloride the least (Table I). This potency scale directly correlates with the ability of these
NHE inhibitors to block NHE activity (Kleyman and Cragoe, 1988, 1990
). We can exclude the possibility that the
reduction of chloroquine accumulation by amilorides results from their properties as weak bases, as there is no
correlation between their constants of inhibition and their
pKa values (Table I). Instead, the clear structure-function relationship observed provides strong evidence that these
compounds prevent chloroquine uptake through the specific inhibition of the P. falciparum NHE.
Secondly, chloroquine uptake coincides with changes in
the cytoplasmic pH and sodium ion concentration, both of
which are indicative of NHE activity. Upon the addition of
chloroquine, protons move from the parasite cytoplasm
into the host erythrocyte, resulting in an alkalization in the
parasite and an acidification of the host erythrocyte. At the
same time, the parasite's cytoplasmic sodium ion concentration rises, an effect that has also been noted previously
by Lee et al. (1988). The increase in osmotic pressure caused by the influx of sodium ions explains why the parasite starts to swell immediately after the addition of chloroquine (Macomber and Sprinz, 1967
; Warhurst and Hockley, 1967
). As chloroquine uptake, proton efflux, and
sodium ion influx all take place at the same time and, significantly, are all inhibited by EIPA, this would suggest a
common basis for these events, i.e., NHE activity. On the
basis of these data we propose that the P. falciparum NHE takes up chloroquine during a sodium/proton exchange
reaction, although the mechanistic details remain to be
determined. The data presented are inconsistent with a
diffusion-controlled model of chloroquine uptake, as this
model can explain neither the acidification of the host erythrocyte cytoplasm nor the sodium influx into the parasite
on the addition of chloroquine.
A quantification of the chloroquine uptake reaction revealed that ~40 mM of sodium ions (from Fig. 7) and 20 mM
of protons were exchanged (from Fig. 4 D), whereas chloroquine was taken up to a concentration of 40 µM (from
Fig. 6) by the CQS parasite clone HB3 in reaching a new
steady-state equilibrium in the presence of chloroquine.
Thus, the stoichiometry of the chloroquine-induced exchange reaction appears to be two sodium ions for one
proton. The P. falciparum chloroquine importer is reminiscent, therefore, of electrogenic NHEs found in invertebrate epithelial cells (Ahearn et al., 1994; Ahearn, 1996
). This
protein is analogous to the vertebrate amiloride-sensitive
electroneutral NHE, except that it performs an extensive
array of transport functions because of its electrogenic nature, transporting a wide range of both monovalent and divalent cations (Ahearn, 1996
). Interestingly, a drug transporting capability has also been demonstrated for an NHE,
where bacterial NHEs export the divalent cation tetracycline in exchange for protons (Yamaguchi et al., 1990
; Cheng
et al., 1996
; Yamaguchi, 1997
). On the basis of these data,
we postulate that the P. falciparum NHE transports the
diprotonated form of chloroquine, which, given its pKa
values of 8.4 and 10.8, respectively, dominates at a physiological pH of 7.3.
The apparent disparity in the amount of chloroquine
taken up in exchange for the huge excess of protons,
would suggest that chloroquine uptake by the P. falciparum NHE is not a simple exchange reaction. We estimate that for each chloroquine molecule taken up, ~500
protons are extruded. It was this disparity that led us to investigate the effect of chloroquine on P. falciparum NHE activity, thereby providing the third line of evidence linking chloroquine uptake with NHE activity. It was found
that chloroquine activates the NHE of the CQS parasite
clone HB3, resulting in an increase in the pH-dependent
activity and working range (Fig. 8). The activation of an
NHE is facilitated by the sodium ion gradient across the
plasma membrane, as shown in other systems (Noel and
Pouyssegur, 1995). Thus, activation of the P. falciparum NHE appears to be required for chloroquine uptake and
accumulation, suggesting that it provides the energy, stored
in the sodium ion gradient across the parasite plasma
membrane, to concentrate chloroquine against its gradient
into the parasite (Fig. 9). Once the NHE has reached its
now activated steady state in the presence of chloroquine,
the surge in ion exchange abates and no more chloroquine is taken up (Figs. 6 and 7). Therefore, chloroquine uptake
appears to be a secondary active transport mechanism in
P. falciparum. This proposal is supported by the observation that CQS parasite clones are rendered insensitive to
chloroquine in the presence of substances, such as monensin, which dissipate the sodium ion gradient (Yayon et al.,
1984
). How chloroquine stimulates the P. falciparum NHE
remains, at present, unknown.
|
CQR parasite clones appear to have preempted most of the stimulatory effect caused by chloroquine, as chloroquine has no significant effect on the NHE activity, as demonstrated for Dd2 (Fig. 8). Its NHE already has an increased pH-dependent activity and working range in the absence of chloroquine, which suggests that the NHEs of CQR parasite clones are constitutively activated (Figs. 5 and 8). As there is no further activation of the NHE of CQR parasites by chloroquine, there is no release in energy; no transient surge of sodium-hydrogen ion exchange occurs and, hence, no chloroquine is concentrated into the parasite (Fig. 9). Based on these data, we propose that the inability of chloroquine to effectively stimulate its own uptake through the constitutively activated NHEs of CQR parasites constitutes a minimal and necessary event in the generation of the CQR phenotype.
Consistent with our proposal that NHE of CQR parasites are altered in response to chloroquine selection, we
have genetically linked both biochemical and physiological properties of the NHE with the CQR phenotype. Biochemical data have demonstrated that a change in the
chloroquine uptake kinetics, reducing the affinity and maximal transport rate, is genetically linked with the CQR phenotype in the cross between HB3 and Dd2 (Sanchez et al.,
1997). Also, the CQR phenotype is genetically linked with
an elevated cytoplasmic pH, a phenotypical marker that is
further associated with all CQR parasites, independent of
their geographic origin. The latter data were derived from
single-cell fluorimetric pHi measurements made on the
parasite still within its host erythrocyte. As such, the cytoplasm of the host erythrocyte would contribute to the total
fluorimetric signal measured. Given that the parasite's cytoplasmic pH is higher than that of its host erythrocyte,
this contribution would result in an underestimation of the
parasite's pH by ~0.03 pH units, as estimated from the
thickness of the host erythrocyte cytoplasm above the parasite (which is between 0 and 0.125 µm according to electronmicroscopic examinations [Rosenthal et al., 1988
; Sam-Yellowe et al., 1988
]), and the fluorescence signals emitted
from the parasite both within and without the host erythrocyte. This estimation is confirmed by cytoplasmic pH determinations made on isolated parasites, which are slightly higher than those made on intact cells, yet maintain the pH
differences between CQS and CQR parasite clones (Table
II). Thus, any differences between CQS and CQR parasites can be solely attributed to differences in the parasites
themselves, rather than their host cell environment. Although we know very little about the ionic and proteinatious environment of the host erythrocyte cytoplasm, we
observed no differences in either the pH or sodium ion
concentration in the absence of chloroquine, regardless of
the parasite's CQR phenotype (data not shown).
A constitutively activated NHE could result from mutations within either the NHE itself or factors modulating
NHE activity, such as kinases, accessory binding proteins,
or Ca2+-calmodulin (Wakabayashi et al., 1994; Noel and
Pouyssegur, 1995
). Therefore, we would propose that either a P. falciparum NHE or a factor regulating NHE
activity resides within the chloroquine resistance locus defined by the genetic cross between HB3 and Dd2 (Wellems et al., 1991
). Interestingly, the primary candidate for
the CQR phenotype mediator, CG2 (Su et al., 1997
), has
features we would predict for an NHE. As is typical for
electrogenic NHEs, CG2 is a putative membrane protein
located at both the plasma membrane and the vacuole
(Kimura et al., 1994
). Furthermore, CG2 also contains a
consensus amiloride binding motif, putative Ca2+ calmodulin binding sites, and the R/K-X-G-R/K-R/K motif found in many of the metabolite proton or sodium ion symporters and the bacterial tetracycline-proton exchangers (Yamaguchi et al., 1990
; Yoshida et al., 1990
; Noel and Pouyssegur, 1995
).
![]() |
Footnotes |
---|
Received for publication 4 February 1997 and in revised form 21 November 1997.
Address all correspondence to Michael Lanzer, Zentrum für Infektionsforschung, Universität Würzburg, Röntgenring 11, D-97070 Würzburg, Germany. Tel.: (49) 93-13-12-151. Fax: (49) 93-13-12-578.We are grateful to H.J. Lang of Hoechst AG (Frankfurt, Germany) for providing us with EIPA and Hoe 370, A. Scherf (Institute Pasteur, Paris, France), D. Walliker (University of Edinburgh, Edinburgh, UK), M.-O. Rojas (Instituto Nacional de Salud, Bogota, Colombia), and A. Cowman (The Walter and Elisa Hall Institute of Medical Research, Melbourne, Victoria, Australia) for P. falciparum clones, and T. Wellems (National Institutes of Health, Bethesda, MD) for allowing us to use the progeny clones from the genetic cross. We thank P. Horrocks for critically reading the manuscript. We thank the Institute of Physiology at the University of Würzburg for allowing us to use the Attofluor ratio imaging system for cytoplasmic pH measurements. Technical assistance was provided by E. Wilken and secretarial support by C. Borde.
This work was supported by grants from the Bundesministerium Für Bildung, Technologie und Forschung, the Deutsche Forschungsgemeinschaft, and the Sander Stiftung.
![]() |
Abbreviations used in this paper |
---|
i, intracellular buffer capacity;
BCECF-AM, fluorochromo 2
7
-bis-(2-carboxyethyl)-5,6-carboxyfluorescein acetomethyl ester;
CQR, chloroquine resistant;
EIPA, 5-(N-ethyl-
N-isopropyl)amiloride;
CQS, chloroquine sensitive;
HMA, 5-N(N-hexamethylene)amiloride;
IC50, 50% inhibitory concentration;
pHi, intracellular pH;
NHE, Na+/H+ exchanger;
SBFI, sodium-binding benzofuran
isophthalate-acetoxymethylester.
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